https://wiki.phagocytes.ca/api.php?action=feedcontributions&feedformat=atom&user=Dicksonb 2026-04-17T11:05:49Z User contributions MediaWiki 1.40.1 https://wiki.phagocytes.ca/index.php?title=Lipisome_and_Lipid-Coated_Beads&diff=218 2023-02-07T22:56:26Z

<p>Dicksonb: Updated protocol. This is the method that Austin used for his experiments and how I was trained to do.</p> <hr /> <div>{{DISPLAYTITLE:Liposome and Lipid-Coated Beads}}<br /> <br /> = General Notes =<br /> <br /> *All buffers should be filtered through a 0.2um filter to remove any particulates before use <br /> *Avoid plastics until beads/lipisomes are generated, as lipids will stick to these <br /> *Clean all equipment toughly with chloroform or 100% EtOH to avoid cross-contamination with lipids <br /> <br /> = Protocol =<br /> '''Updated Protocol for PtdSer Beads (As per Austin Lam and Brandon Dickson)'''<br /> <br /> # Using a glass syringe, add 30 μL PtdChol (10 mg/mL stock), 8 μL PtdSer (10 mg/mL stock), and 8 μL of biotinylated PtdEth (10 mg/mL stock) to a small amber vial. <br /> #* Ensure that you flush the syringe completely with chloroform with chloroform every time before switching vials. Multiple flushes will be required. <br /> #* If using rhodamine-PtdEth, use this last to avoid contaminating the other stocks with red lipid.<br /> #* Flush vials containing the stock solutions with nitrogen gas, then cap and seal with parafilm before returning to freezer. <br /> # Evaporate the lipid mixture in the amber vial with a light stream of nitrogen gas, rotating the vial with your hands. <br /> #* Continue for a few minutes until the vial is completely dry. <br /> # Add 400 μL of PBS into the vial. Vortex vial at max intensity for 1 minute to suspend completely. <br /> # Vortex the small container containing the silica beads. Transfer 100 μL of silica beads into a 1.5 mL tube. Wash with 1 mL ddH&lt;sub&gt;2&lt;/sub&gt;O at 5000 x g for 1 minute. Carefully remove supernatant. Repeat for a total of three washes. <br /> #* Remove as much water as possible after the last wash. <br /> # Resuspend the beads in 400 μL of the lipid solution. Mix by pipetting.<br /> # Rotate the bead-lipid mix at room temperature for 20 minutes using the rotisserie-style spinner.<br /> #* Cover tube with tin foil if using rhodamine-PtdEth to avoid photobleaching<br /> # Spin the beads down at 5000 x g for 30 seconds. Remove supernatant carefully. <br /> #* This step removes unincorporated liposomes<br /> # Resuspend beads in 400 μL of PBS. Briefly purge the tube with nitrogen gas before capping. Store at 4°C for up to 4 days. <br /> <br /> ===== Lipid Preparation: =====<br /> <br /> #Using the lipid calculation spreadsheet, calculate the amount of lipids required. <br /> #*If using dansylated lipids, add at 2-4% total molar amount. Higher concentrations will self-quench.&lt;br&gt; <br /> #*Generally speaking, 4μmol of lipids will produce enough for 1 weeks work&lt;br&gt; <br /> #*Once made, lipids are good for 3-4 days at 4oC&lt;br&gt; <br /> <br /> *'''In a hood:'''<br /> <br /> #&lt;li value=&quot;2&quot;&gt;Clean glass syringes 5x with choloform.&lt;/li&gt; <br /> #Using the syringes, transfer the desired amount of lipids from the stock vials into small glass tubes. Do not use plastic for any step.&lt;br&gt; <br /> #Between each lipid, clean syringe by washing 5x with choloroform.&lt;br&gt; <br /> #Before sealing each stock tube, fill tube with N2 or CO2 to prevent lipid oxidation&lt;br&gt; <br /> #Dry the lipids to the lower ¼ of the tube by placing the tube at an ~45o angle, and rotating the tube while gently flowing N2 or CO2 into the tube.&lt;br&gt; <br /> #Once the tubes are dried, place tubes beneath tube drier, and dry for an additional 2hrs-over night using N2 of CO2. Protect tubes from light during this time.&lt;br&gt; <br /> #During step 7, clean syringes 5-10x in chloroform and return to storage area.&lt;br&gt;<br /> <br /> ===== Lipisome Preparation:&lt;br&gt; =====<br /> <br /> #To the dried lipids add 400μl of desired buffer (i.e. 20mM HEPES, pH 7.2)&lt;br&gt; <br /> #Vortex each tube until as much white precipitate goes into solution as possible. Some lipids will not resuspend well (i.e. PC, PE)&lt;br&gt; <br /> #Sonicate tubes 2-3x, 30sec, in ice-cold water, using an immersion sonicator at 30-35% maximum power.&lt;br&gt; <br /> #Clean syringes for lipisome extruder in 100% EtOH and assemble.&lt;br&gt; <br /> #Remove o-rings and mesh from extruders. Using syringes, pass 100% EtOH through each half of the extruder 5x, then the desired buffer 1x.&lt;br&gt; <br /> #Wash o-rings in ddH2O and place back into extruder. Using a vacuum, remove any excess fluid from o-rings and ports.&lt;br&gt; <br /> #Place 2 100nm-pore filters onto one o-ring. Filters should be centered on extruder, with an equal amount extending over o-rings on each side.&lt;br&gt; <br /> #Insert into metal casing, followed by second half of extruder. Screw caps on tightly; while tightening watch filters through observation port to ensure they are sealed properly.&lt;br&gt; <br /> #Place a long needle (i.e. 1.5” 18g) onto one glass syringe. Suck up lipid into syringe, making sure no air bubbles are trapped. If necessary lipids can be transferred into plastic eppindorfs if glass tubes are too long for needles.&lt;br&gt; <br /> #Insert sample-containing syringe onto the short port of the extruder. Attach the empty syringe to the long-port. Press solution through extruder 30 times; if the solution remains turbid at this point pass through additional times until solution becomes clear. Collect purified lipisomes into eppindorf tubes, using the syringe on the long port to ensure maximum recovery and prevent contamination with unprocessed sample.&lt;br&gt; <br /> #Disassemble extruder, throw away 100nm filters, and clean the extruder as described in steps 4-6 between each sample.&lt;br&gt; <br /> #When complete clean extruder with EtOH as described above. When finished, reassemble extruder, with o-rings, and pass 100% EtOH through system 5 times. Disassemble completely and place in carrying case.&lt;br&gt; <br /> #Liposomes can be stored at 4oC for 3-4 days.&lt;br&gt; <br /> <br /> ===== Lipid-Coated Bead Preparation: =====<br /> <br /> #Dissassemble HPLC column and collect nucleosil beads into a lipid-free container. Store at -20oC until needed. For each lipid sample measure out 2mg beads. Dilute into 100μl chloroform per 2mg beads and sonicate. <br /> #Before step 6 of the lipid preparation (first drying of the aliquotted lipids) add 100μl of the bead/chloroform mixture. Once added, dry the lipid/bead preparation protocol as per usual. <br /> #Resuspend dried lipids in 1.5ml of desired buffer. Vortex and sonicate as described in lipisome section to suspend. <br /> #Transfer to 1.5ml eppidorf tubes by inverting glass tube overtop of eppidorf. Vortex in minifuge, 4000rpm, 4min. <br /> #Remove supernatant, resuspend in 1ml buffer, and spin at 6000 RPM for 4 min. Repeat 2-3x <br /> #Put an 8μl spot on a slide and cover with cover slip. View under microscope; if beads are clumped further processing is required; otherwise go to step 7. <br /> #If sample is still clumped, spin 5min at 6000 RPM&lt;br&gt; <br /> #*Remove supernatant&lt;br&gt; <br /> #*Gently profuse buffer across pellet to wash&lt;br&gt; <br /> #*Remove wash and resuspend&lt;br&gt; <br /> #*Sonciate in immersion sonicator, 30sec at 30% maximum power&lt;br&gt; <br /> #*Repeat until clumps are gone <br /> #Spin down cleaned sample at 4500RPM, 4min, resuspend in 1ml clean buffer, and count beads on scope using a hematocytometer and 100x magnification. Normalize bead concentration and store beads at 4oC until needed. Beads will last 3-4 days at 4oC.&lt;br&gt;</div>

Dicksonb https://wiki.phagocytes.ca/index.php?title=Bradford_Assay&diff=214 2022-05-24T14:06:36Z

<p>Dicksonb: </p> <hr /> <div>'''Introduction'''<br /> <br /> The Bradford assay should be used to quantify total protein within a sample relative to a standard curve. This is important when running proteins on SDS-PAGE/Western blot, where each lane must contain an identical amount of total protein. This way, the intensity of the bands can be compared between different lanes, as standardizing the amount of protein loaded per lane will account for any variation in cell count from which the proteins were derived. <br /> <br /> '''Protocol'''<br /> <br /> ''For a 96 well plate''<br /> <br /> # Using a 0.2 or 0.45 μm filter and 15 mL syringe, filter Bio-Rad Protein Assay Dye Reagent Concentrate (Bradford Dye) into a 15 mL falcon tube. Dilute 1:5 with ddH2O (1 part concentrate, 4 parts ddH2O). Invert to mix. Let warm to room temperature. <br /> # Collect lysate or supernatant containing protein. Keep on ice.<br /> # Label eight 1.5 mL tubes as: 1, 2, 3, 4, 5, 6, 7, 8. Set up the BSA standard by following '''Table 1'''. (Use the Bio-Rad 2 mg/mL stock. Do not make BSA yourself)<br /> #*Make sure the diluent is the same as buffer that the protein of interest is in. For example, if in PBS, use PBS to dilute the standard. <br /> #*Mix by pipetting up and down. Avoid creating bubbles in the tube <br /> #Set up 500 μL dilutions of your sample of interest. Make dilutions of 1:5, 1:10, and 1:25. <br /> #*If you think your sample is very concentrated, instead make 1:10, 1:25, and 1:25 dilutions <br /> #*If you think your sample is very dilute, use undiluted, 1:2, and 1:5 dilutions. (This uses a lot of sample however!) <br /> #Add 150 μL of sample or standard to each well following '''Figure 1'''. Avoid bubbles by only pipetting to the first stop.<br /> # Pour the dye into a multichannel pipette reservoir. Using a multichannel pipette, pipet 150 μL of dye into each well. Mix by pipetting carefully up and down.<br /> # Incubate for at least 5 minutes at room temperature. Do not exceed 1 hr. <br /> # Take off the lid of the plate, and place it into a plate reader/spectrometer.<br /> # Measure absorbance at 595 nm.<br /> {| class=&quot;wikitable&quot;<br /> |+Table 1.<br /> !Tube #<br /> !Standard volume (μL)<br /> !Source of standard<br /> !Diluent volume (μL)<br /> !Final concentration (μg/mL)<br /> |-<br /> |1<br /> |10<br /> |2 mg/mL stock<br /> |790<br /> |25<br /> |-<br /> |2<br /> |10<br /> |2 mg/mL stock<br /> |990<br /> |20<br /> |-<br /> |3<br /> |6<br /> |2 mg/mL stock<br /> |794<br /> |15<br /> |-<br /> |4<br /> |500<br /> |Tube 2<br /> |500<br /> |10<br /> |-<br /> |5<br /> |500<br /> |Tube 4<br /> |500<br /> |5<br /> |-<br /> |6<br /> |500<br /> |Tube 5<br /> |500<br /> |2.5<br /> |-<br /> |7<br /> |500<br /> |Tube 6<br /> |500<br /> |1.25<br /> |-<br /> |8<br /> | -<br /> | -<br /> |500<br /> |0<br /> |}<br /> [[File:96 well plate for wiki (1).png|left|thumb|500x500px|'''Figure 1''']]<br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> -<br /> <br /> <br /> ''Analysis using the standard curve''<br /> <br /> # Export results from spectrometer to Excel.<br /> # Subtract the average absorbance of each standard/sample by the average absorbance of the blank. Essentially, you are setting the blank absorption to 0 and making all readings relative to that.<br /> # Plot the known standard concentrations (X-axis: 0, 1.25, 2.5, 5, 10, 15, 20, and 25 μg/mL) with the average absorbance at each concentration (Y-axis). '''Add a line of best fit and display the equation with R&lt;sup&gt;2&lt;/sup&gt; value.''' Ideally, the R&lt;sup&gt;2&lt;/sup&gt; value should be ≥ 0.99. R&lt;sup&gt;2&lt;/sup&gt; &lt; 0.94 curves should not be used. <br /> # Calculate the average absorbance for the samples of interest by using the equation of the line graph y = mx+b (Absorbance = Slope*Concentration + b) and solve for x.<br /> # If the calculated concentration falls within the linear range of the standard curve (1.25 - 25 μg/mL), then it is accurate. Determine the concentration of the undiluted sample by multiplying by the dilution factor.<br /> <br /> # <br /> <br /> #</div>

Dicksonb https://wiki.phagocytes.ca/index.php?title=Bradford_Assay&diff=165 2022-02-27T13:49:08Z

<p>Dicksonb: </p> <hr /> <div>'''Introduction'''<br /> <br /> The Bradford assay should be used to quantify total protein within a sample relative to a standard curve. This is important when running proteins on SDS-PAGE/Western blot, where each lane must contain an identical amount of total protein. This way, the intensity of the bands can be compared between different lanes, as standardizing the amount of protein loaded per lane will account for any variation in cell count from which the proteins were derived. <br /> <br /> '''Protocol'''<br /> <br /> ''For a 96 well plate''<br /> <br /> # Using a 0.2 or 0.45 μm filter and 15 mL syringe, filter Bio-Rad Protein Assay Dye Reagent Concentrate (Bradford Dye) into a 15 mL falcon tube. Dilute 1:5 with ddH2O (1 part concentrate, 4 parts ddH2O). Invert to mix. Let warm to room temperature. <br /> # Collect lysate or supernatant containing protein. Keep on ice.<br /> # Label eight 1.5 mL tubes as: 1, 2, 3, 4, 5, 6, 7, 8. Set up the BSA standard by following '''Table 1'''. (Use the Bio-Rad 2 mg/mL stock. Do not make BSA yourself)<br /> #*Make sure the diluent is the same as buffer that the protein of interest is in. For example, if in PBS, use PBS to dilute the standard. If in RIPA buffer, use RIPA, etc. <br /> #*Mix by pipetting up and down. Avoid creating bubbles in the tube <br /> #Set up 500 μL dilutions of your sample of interest. Make dilutions of 1:5, 1:10, and 1:25. <br /> #*If you think your sample is very concentrated, instead make 1:10, 1:25, and 1:25 dilutions <br /> #*If you think your sample is very dilute, use undiluted, 1:2, and 1:5 dilutions. (This uses a lot of sample however!) <br /> #Add 150 μL of sample or standard to each well following '''Figure 1'''. Avoid bubbles by only pipetting to the first stop.<br /> # Pour the dye into a multichannel pipette reservoir. Using a multichannel pipette, pipet 150 μL of dye into each well. Mix by pipetting carefully up and down.<br /> # Incubate for at least 5 minutes at room temperature. Do not exceed 1 hr. <br /> # Take off the lid of the plate, and place it into a plate reader/spectrometer.<br /> # Measure absorbance at 595 nm.<br /> {| class=&quot;wikitable&quot;<br /> |+Table 1.<br /> !Tube #<br /> !Standard volume (μL)<br /> !Source of standard<br /> !Diluent volume (μL)<br /> !Final concentration (μg/mL)<br /> |-<br /> |1<br /> |10<br /> |2 mg/mL stock<br /> |790<br /> |25<br /> |-<br /> |2<br /> |10<br /> |2 mg/mL stock<br /> |990<br /> |20<br /> |-<br /> |3<br /> |6<br /> |2 mg/mL stock<br /> |794<br /> |15<br /> |-<br /> |4<br /> |500<br /> |Tube 2<br /> |500<br /> |10<br /> |-<br /> |5<br /> |500<br /> |Tube 4<br /> |500<br /> |5<br /> |-<br /> |6<br /> |500<br /> |Tube 5<br /> |500<br /> |2.5<br /> |-<br /> |7<br /> |500<br /> |Tube 6<br /> |500<br /> |1.25<br /> |-<br /> |8<br /> | -<br /> | -<br /> |500<br /> |0<br /> |}<br /> [[File:96 well plate for wiki (1).png|left|thumb|500x500px|'''Figure 1''']]<br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> -<br /> <br /> <br /> ''Analysis using the standard curve''<br /> <br /> # Export results from spectrometer to Excel.<br /> # Subtract the average absorbance of each standard/sample by the average absorbance of the blank. Essentially, you are setting the blank absorption to 0 and making all readings relative to that.<br /> # Plot the known standard concentrations (X-axis: 0, 1.25, 2.5, 5, 10, 15, 20, and 25 μg/mL) with the average absorbance at each concentration (Y-axis). '''Add a line of best fit and display the equation with R&lt;sup&gt;2&lt;/sup&gt; value.''' Ideally, the R&lt;sup&gt;2&lt;/sup&gt; value should be ≥ 0.99. R&lt;sup&gt;2&lt;/sup&gt; &lt; 0.94 curves should not be used. <br /> # Calculate the average absorbance for the samples of interest by using the equation of the line graph y = mx+b (Absorbance = Slope*Concentration + b) and solve for x.<br /> # If the calculated concentration falls within the linear range of the standard curve (1.25 - 25 μg/mL), then it is accurate. Determine the concentration of the undiluted sample by multiplying by the dilution factor.<br /> <br /> # <br /> <br /> #</div>

Dicksonb https://wiki.phagocytes.ca/index.php?title=Bradford_Assay&diff=164 2022-02-27T13:47:46Z

<p>Dicksonb: Huge edit to fix the previous protocol, which was completely wrong.</p> <hr /> <div>'''Introduction'''<br /> <br /> The Bradford assay should be used to quantify total protein within a sample relative to a standard curve. This is important when running proteins on SDS-PAGE/Western blot, where each lane must contain an identical amount of total protein. This way, the intensity of the bands can be compared between different lanes, as standardizing the amount of protein loaded per lane will account for any variation in cell count from which the proteins were derived. <br /> <br /> '''Protocol'''<br /> <br /> ''For a 96 well plate''<br /> <br /> # Using a 0.2 or 0.45 μm filter and 15 mL syringe, filter Bio-Rad Protein Assay Dye Reagent Concentrate (Bradford Dye) into a 15 mL falcon tube. Dilute 1:5 with ddH2O (1 part concentrate, 4 parts ddH2O). Invert to mix. Let warm to room temperature. <br /> # Collect lysate or supernatant containing protein. Keep on ice.<br /> # Label eight 1.5 mL tubes as: 1, 2, 3, 4, 5, 6, 7, 8. Set up the BSA standard by following '''Table 1'''. (Use the Bio-Rad 2 mg/mL stock. Do not make BSA yourself)<br /> #*Make sure the diluent is the same as buffer that the protein of interest is in. For example, if in PBS, use PBS to dilute the standard. If in RIPA buffer, use RIPA, etc. <br /> #*Mix by pipetting up and down. Avoid creating bubbles in the tube <br /> #Set up 500 μL dilutions of your sample of interest. Make dilutions of 1:5, 1:10, and 1:25. <br /> #*If you think your sample is very concentrated, instead make 1:10, 1:25, and 1:25 dilutions <br /> #*If you think your sample is very dilute, use undiluted, 1:2, and 1:5 dilutions. (This uses a lot of sample however!) <br /> #Add 150 μL of sample or standard to each well following '''Figure 1'''. Avoid bubbles by only pipetting to the first stop.<br /> # Pour the dye into a multichannel pipette reservoir. Using a multichannel pipette, pipet 150 μL of dye into each well. Mix by pipetting carefully up and down.<br /> # Incubate for at least 5 minutes at room temperature. Do not exceed 1 hr. <br /> # Take off the lid of the plate, and place it into a plate reader/spectrometer.<br /> # Measure absorbance at 595 nm.<br /> {| class=&quot;wikitable&quot;<br /> |+Table 1.<br /> !Tube #<br /> !Standard volume (μL)<br /> !Source of standard<br /> !Diluent volume (μL)<br /> !Final concentration (μg/mL)<br /> |-<br /> |1<br /> |10<br /> |2 mg/mL stock<br /> |790<br /> |25<br /> |-<br /> |2<br /> |10<br /> |2 mg/mL stock<br /> |990<br /> |20<br /> |-<br /> |3<br /> |6<br /> |2 mg/mL stock<br /> |794<br /> |15<br /> |-<br /> |4<br /> |500<br /> |Tube 2<br /> |500<br /> |10<br /> |-<br /> |5<br /> |500<br /> |Tube 4<br /> |500<br /> |5<br /> |-<br /> |6<br /> |500<br /> |Tube 5<br /> |500<br /> |2.5<br /> |-<br /> |7<br /> |500<br /> |Tube 6<br /> |500<br /> |1.25<br /> |-<br /> |8<br /> | -<br /> | -<br /> |500<br /> |0<br /> |}<br /> [[File:96 well plate for wiki (1).png|left|thumb|500x500px|'''Figure 1''']]<br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> ''Analysis using the standard curve''<br /> <br /> # Export results from spectrometer to Excel.<br /> # Subtract the average absorbance of each standard/sample by the average absorbance of the blank. Essentially, you are setting the blank absorption to 0 and making all readings relative to that.<br /> # Plot the known standard concentrations (X-axis: 0, 1.25, 2.5, 5, 10, 15, 20, and 25 μg/mL) with the average absorbance at each concentration (Y-axis). '''Add a line of best fit and display the equation with R&lt;sup&gt;2&lt;/sup&gt; value.''' Ideally, the R&lt;sup&gt;2&lt;/sup&gt; value should be ≥ 0.99. R&lt;sup&gt;2&lt;/sup&gt; &lt; 0.94 curves should not be used. <br /> # Calculate the average absorbance for the samples of interest by using the equation of the line graph y = mx+b (Absorbance = Slope*Concentration + b) and solve for x.<br /> # If the calculated concentration falls within the linear range of the standard curve (1.25 - 25 μg/mL), then it is accurate. Determine the concentration of the undiluted sample by multiplying by the dilution factor.<br /> <br /> # <br /> <br /> #</div>

Dicksonb https://wiki.phagocytes.ca/index.php?title=File:96_well_plate_for_wiki_(1).png&diff=163 2022-02-27T13:44:32Z

<p>Dicksonb: </p> <hr /> <div>For the microassay</div>

Dicksonb https://wiki.phagocytes.ca/index.php?title=Bradford_Assay&diff=152 2022-02-17T23:26:24Z

<p>Dicksonb: </p> <hr /> <div>'''Introduction'''<br /> <br /> The Bradford assay should be used to quantify total protein within a sample relative to a standard curve. This is important when running proteins on SDS-PAGE/Western blot, where each lane must contain an identical amount of total protein. This way, the intensity of the bands can be compared between different lanes, as standardizing the amount of protein loaded per lane will account for any variation in cell count from which the proteins were derived. <br /> '''Protocol'''<br /> <br /> '''Protocol'''<br /> <br /> ''Some things to know'':<br /> <br /> * This protocol assumes the use of bovine serum albumin (BSA) for the standard curve, which has a linear range of 200 μg/mL to 1000 μg/mL within Bio-Rad's protein assay dye. <br /> * If your protein is within a supernatant containing an acid indicator, such as phenol red, this may interfere with the assay. If your protein is within a solution containing phenol red, then ensure that all samples, standards, and blanks are diluted with that media (e.g. Serum free DMEM).<br /> * Each tube will have extra volume to ensure you have enough for the last replicate and to avoid air bubbles.<br /> * Reverse pipetting technique is highly recommended to avoid air bubbles and to ensure accuracy. <br /> ''For a 96 well plate''<br /> <br /> # Using a 0.2 μm filter and 15 mL syringe, filter Bio-Rad Protein Assay Dye Reagent Concentrate (Bradford Dye) into a 1.5 mL Eppendorf tube or 5 mL tube. Let warm to room temperature. <br /> # Collect lysate or supernatant containing protein. Keep on ice.<br /> # In separate 1.5 mL tubes, dilute the sample using ddH2O (or whatever buffer/media is appropriate) into 1:2 and 1:10 dilutions. <br /> # Prepare a 10 mg/mL stock solution of BSA by dissolving BSA in ddH2O. Prepare a dilution series using ddH2O (or whatever buffer/media is appropriate) in 1.5 mL tubes: 1 mg/mL, 0.8 mg/mL, 0.6 mg/mL, 0.4 mg/mL, and 0.2 mg/mL. <br /> # See '''Figure 1''' for a general plate setup. 80 μL of sample and standard will be used per well. <br /> # Spin down the plate at 300 rpm for 1 min to remove bubbles and any liquid on the side of the well.<br /> # Pour filtered Bradford Dye into a multichannel pipette reservoir. Using a multichannel pipette, pipet dye into each well such that the dye is diluted 1/5 (for example, if 80 μL of sample was loaded per well, then 20 μL of dye will be added in each well) If a multichannel pipette is not available, use the micropipette and pipet into each well '''as quickly as possible'''. <br /> # Incubate for at least 5 minutes at room temperature. Do not exceed 1 hr. <br /> # Take off the lid of the plate, and place it into a plate reader/spectrometer.<br /> # Measure absorbance at 595 nm.<br /> [[File:Bradford plate layout.png|thumb|'''Figure 1.''' General plate layout |border|center]]<br /> <br /> <br /> <br /> ''Analysis using the standard curve''<br /> <br /> # Export results from spectrometer to Excel.<br /> # Subtract the average absorbance of each well by the average absorbance of the blank.<br /> # Plot the known standard concentrations (X-axis; 0.2, 0.4, 0.6, 0.8, and 1.0 mg/mL) with the average absorbance at each concentration (Y-axis). '''Add a line of best fit and display the equation with R&lt;sup&gt;2&lt;/sup&gt; value.''' Ideally, the R&lt;sup&gt;2&lt;/sup&gt; value should be ≥ 0.99. R&lt;sup&gt;2&lt;/sup&gt; &lt; 0.94 curves should not be used. <br /> # Calculate the average absorbance for the samples of interest by using the equation of the line graph y = mx+b (Absorbance = Slope*Concentration + b) and solve for x.<br /> # If the calculated concentration falls within the linear range of the standard curve (0.2 - 1.0 mg/mL), then it is accurate. If necessary, determine the concentration of the undiluted sample by multiplying by the dilution factor. <br /> <br /> # <br /> <br /> #</div>

Dicksonb https://wiki.phagocytes.ca/index.php?title=Lipisome_and_Lipid-Coated_Beads&diff=140 2022-01-15T14:27:40Z

<p>Dicksonb: </p> <hr /> <div>{{DISPLAYTITLE:Liposome and Lipid-Coated Beads}}<br /> <br /> = General Notes =<br /> <br /> *All buffers should be filtered through a 0.2um filter to remove any particulates before use <br /> *Avoid plastics until beads/lipisomes are generated, as lipids will stick to these <br /> *Clean all equipment toughly with chloroform or 100% EtOH to avoid cross-contamination with lipids <br /> <br /> = Protocol =<br /> <br /> ===== Lipid Preparation: =====<br /> <br /> #Using the lipid calculation spreadsheet, calculate the amount of lipids required. <br /> #*If using dansylated lipids, add at 2-4% total molar amount. Higher concentrations will self-quench.&lt;br&gt; <br /> #*Generally speaking, 4μmol of lipids will produce enough for 1 weeks work&lt;br&gt; <br /> #*Once made, lipids are good for 3-4 days at 4oC&lt;br&gt; <br /> <br /> *'''In a hood:'''<br /> <br /> #&lt;li value=&quot;2&quot;&gt;Clean glass syringes 5x with choloform.&lt;/li&gt; <br /> #Using the syringes, transfer the desired amount of lipids from the stock vials into small glass tubes. Do not use plastic for any step.&lt;br&gt; <br /> #Between each lipid, clean syringe by washing 5x with choloroform.&lt;br&gt; <br /> #Before sealing each stock tube, fill tube with N2 or CO2 to prevent lipid oxidation&lt;br&gt; <br /> #Dry the lipids to the lower ¼ of the tube by placing the tube at an ~45o angle, and rotating the tube while gently flowing N2 or CO2 into the tube.&lt;br&gt; <br /> #Once the tubes are dried, place tubes beneath tube drier, and dry for an additional 2hrs-over night using N2 of CO2. Protect tubes from light during this time.&lt;br&gt; <br /> #During step 7, clean syringes 5-10x in chloroform and return to storage area.&lt;br&gt;<br /> <br /> ===== Lipisome Preparation:&lt;br&gt; =====<br /> <br /> #To the dried lipids add 400μl of desired buffer (i.e. 20mM HEPES, pH 7.2)&lt;br&gt; <br /> #Vortex each tube until as much white precipitate goes into solution as possible. Some lipids will not resuspend well (i.e. PC, PE)&lt;br&gt; <br /> #Sonicate tubes 2-3x, 30sec, in ice-cold water, using an immersion sonicator at 30-35% maximum power.&lt;br&gt; <br /> #Clean syringes for lipisome extruder in 100% EtOH and assemble.&lt;br&gt; <br /> #Remove o-rings and mesh from extruders. Using syringes, pass 100% EtOH through each half of the extruder 5x, then the desired buffer 1x.&lt;br&gt; <br /> #Wash o-rings in ddH2O and place back into extruder. Using a vacuum, remove any excess fluid from o-rings and ports.&lt;br&gt; <br /> #Place 2 100nm-pore filters onto one o-ring. Filters should be centered on extruder, with an equal amount extending over o-rings on each side.&lt;br&gt; <br /> #Insert into metal casing, followed by second half of extruder. Screw caps on tightly; while tightening watch filters through observation port to ensure they are sealed properly.&lt;br&gt; <br /> #Place a long needle (i.e. 1.5” 18g) onto one glass syringe. Suck up lipid into syringe, making sure no air bubbles are trapped. If necessary lipids can be transferred into plastic eppindorfs if glass tubes are too long for needles.&lt;br&gt; <br /> #Insert sample-containing syringe onto the short port of the extruder. Attach the empty syringe to the long-port. Press solution through extruder 30 times; if the solution remains turbid at this point pass through additional times until solution becomes clear. Collect purified lipisomes into eppindorf tubes, using the syringe on the long port to ensure maximum recovery and prevent contamination with unprocessed sample.&lt;br&gt; <br /> #Disassemble extruder, throw away 100nm filters, and clean the extruder as described in steps 4-6 between each sample.&lt;br&gt; <br /> #When complete clean extruder with EtOH as described above. When finished, reassemble extruder, with o-rings, and pass 100% EtOH through system 5 times. Disassemble completely and place in carrying case.&lt;br&gt; <br /> #Liposomes can be stored at 4oC for 3-4 days.&lt;br&gt; <br /> <br /> ===== Lipid-Coated Bead Preparation: =====<br /> <br /> #Dissassemble HPLC column and collect nucleosil beads into a lipid-free container. Store at -20oC until needed. For each lipid sample measure out 2mg beads. Dilute into 100μl chloroform per 2mg beads and sonicate. <br /> #Before step 6 of the lipid preparation (first drying of the aliquotted lipids) add 100μl of the bead/chloroform mixture. Once added, dry the lipid/bead preparation protocol as per usual. <br /> #Resuspend dried lipids in 1.5ml of desired buffer. Vortex and sonicate as described in lipisome section to suspend. <br /> #Transfer to 1.5ml eppidorf tubes by inverting glass tube overtop of eppidorf. Vortex in minifuge, 4000rpm, 4min. <br /> #Remove supernatant, resuspend in 1ml buffer, and spin at 6000 RPM for 4 min. Repeat 2-3x <br /> #Put an 8μl spot on a slide and cover with cover slip. View under microscope; if beads are clumped further processing is required; otherwise go to step 7. <br /> #If sample is still clumped, spin 5min at 6000 RPM&lt;br&gt; <br /> #*Remove supernatant&lt;br&gt; <br /> #*Gently profuse buffer across pellet to wash&lt;br&gt; <br /> #*Remove wash and resuspend&lt;br&gt; <br /> #*Sonciate in immersion sonicator, 30sec at 30% maximum power&lt;br&gt; <br /> #*Repeat until clumps are gone <br /> #Spin down cleaned sample at 4500RPM, 4min, resuspend in 1ml clean buffer, and count beads on scope using a hematocytometer and 100x magnification. Normalize bead concentration and store beads at 4oC until needed. Beads will last 3-4 days at 4oC.&lt;br&gt;</div>

Dicksonb https://wiki.phagocytes.ca/index.php?title=Bradford_Assay&diff=139 2022-01-15T14:23:51Z

<p>Dicksonb: </p> <hr /> <div>'''Introduction'''<br /> <br /> The Bradford assay should be used to quantify total protein within a sample relative to a standard curve. This is important when running proteins on SDS-PAGE/Western blot, where each lane must contain an identical amount of total protein. This way, the intensity of the bands can be compared between different lanes, as standardizing the amount of protein loaded per lane will account for any variation in cell count from which the proteins were derived. <br /> <br /> '''Protocol'''<br /> <br /> ''Some things to know'':<br /> <br /> * This protocol assumes the use of bovine serum albumin (BSA) for the standard curve, which has a linear range of 200 μg/mL to 1000 μg/mL within Bio-Rad's protein assay dye. <br /> * If your protein is within a supernatant containing an acid indicator, such as phenol red, this may interfere with the assay. If your protein is within a solution containing phenol red, then ensure that all samples, standards, and blanks are diluted with that media (e.g. Serum free DMEM).<br /> * Each tube will have extra volume to ensure you have enough for the last replicate and to avoid air bubbles.<br /> * Reverse pipetting technique is highly recommended to avoid air bubbles and to ensure accuracy. <br /> ''For a 96 well plate''<br /> <br /> # Using a 0.2 μm filter and 15 mL syringe, filter approximately 4 mL of Bio-Rad Protein Assay Dye Reagent Concentrate (Bradford Dye) into a 5 mL Eppendorf tube or 15 mL falcon tube. Let it warm to room temperature. <br /> # Collect lysate or supernatant that which the protein concentration needs to be calculated for. Keep on ice. <br /> # In separate 1.5 mL tubes, dilute the sample using ddH2O (or whatever buffer/media is appropriate) into 1:2 and 1:10 dilutions into a total of 600 μL. ('''NOTE:''' Do not use the 1:2 diluted sample to prepare the 1:10 tube. Use the original, undiluted sample to prepare the 1:10 dilution)<br /> # Prepare a 1 mg/mL stock solution of BSA by dissolving BSA in ddH2O. Prepare a dilution series using ddH2O (or whatever buffer/media is appropriate) in 1.5 mL tubes: 1 mg/mL, 0.8 mg/mL, 0.6 mg/mL, 0.4 mg/mL, and 0.2 mg/mL.<br /> # See '''Figure 1''' for a general plate setup. Using the reverse pipetting technique, pipet 160 μL of blank, standard, and samples into each appropriate well. Avoid creating air bubbles.<br /> # Spin down the plate at 300 rpm for 1 min to remove bubbles and any liquid on the side of the well.<br /> # Dump 4 mL of filtered Bradford Dye into a multichannel pipette reservoir. Using a multichannel pipette, pipet 40 μL of dye into each well. If a multichannel pipette is not available, '''''quickly''''' pipet 40 μL of dye into each well using a P200 pipette. <br /> # Incubate for at least 5 minutes at room temperature. Do not exceed 1 hr. <br /> # Take off the lid of the plate, and place it into a plate reader/spectrometer.<br /> # Measure absorbance at 595 nm.<br /> [[File:Bradford plate layout.png|thumb|'''Figure 1.''' General plate layout |border|center]]<br /> <br /> <br /> <br /> ''Analysis using the standard curve''<br /> <br /> # Export results from spectrometer to Excel.<br /> # Subtract the average absorbance of each well by the average absorbance of the blank.<br /> # Plot the known standard concentrations (X-axis; 0.2, 0.4, 0.6, 0.8, and 1.0 mg/mL) with the average absorbance at each concentration (Y-axis). '''Add a line of best fit and display the equation with R&lt;sup&gt;2&lt;/sup&gt; value.''' Ideally, the R&lt;sup&gt;2&lt;/sup&gt; value should be ≥ 0.99. R&lt;sup&gt;2&lt;/sup&gt; &lt; 0.94 curves should not be used. <br /> # Calculate the average absorbance for the samples of interest by using the equation of the line graph y = mx+b (Absorbance = Slope*Concentration + b) and solve for x.<br /> # If the calculated concentration falls within the linear range of the standard curve (0.2 - 1.0 mg/mL), then it is accurate. If necessary, determine the concentration of the undiluted sample by multiplying by the dilution factor. <br /> <br /> # <br /> <br /> #</div>

Dicksonb https://wiki.phagocytes.ca/index.php?title=Bradford_Assay&diff=138 2022-01-15T14:21:41Z

<p>Dicksonb: </p> <hr /> <div>'''Introduction'''<br /> <br /> The Bradford assay should be used to quantify total protein within a sample relative to a standard curve. This is important when running proteins on SDS-PAGE/Western blot, where each lane must contain an identical amount of total protein. This way, the intensity of the bands can be compared between different lanes, as standardizing the amount of protein loaded per lane will account for any variation in cell count from which the proteins were derived. <br /> <br /> '''Protocol'''<br /> <br /> ''Some things to know'':<br /> <br /> * This protocol uses Bio-Rad products<br /> * This protocol assumes the use of bovine serum albumin (BSA) for the standard curve, which has a linear range of 200 μg/mL to 1000 μg/mL within Bio-Rad's protein assay dye. Linear range may be improved by using other products, but are more expensive than Bio-Rad's. <br /> * If your protein is within a supernatant containing an acid indicator, such as phenol red, this may interfere with the assay. If your protein is within a solution containing phenol red, then ensure that all samples, standards, and blanks are diluted with that media (e.g. Serum free DMEM).<br /> * This protocol assumes all standards, samples, and blanks are run in triplicates.<br /> * Each tube will have extra volume to ensure you have enough for the last replicate. <br /> * Reverse pipetting technique is highly recommended to avoid air bubbles and to ensure accuracy. <br /> * The Bradford Dye is a 5X stock solution.<br /> <br /> ''For a 96 well plate''<br /> <br /> # Using a 0.2 μm filter and 15 mL syringe, filter approximately 4 mL of Bio-Rad Protein Assay Dye Reagent Concentrate (Bradford Dye) into a 5 mL Eppendorf tube or 15 mL falcon tube. Let it warm to room temperature. <br /> # Collect lysate or supernatant that which the protein concentration needs to be calculated for. Keep on ice. <br /> # In separate 1.5 mL tubes, dilute the sample using ddH2O (or whatever buffer/media is appropriate) into 1:2 and 1:10 dilutions into a total of 600 μL. ('''NOTE:''' Do not use the 1:2 diluted sample to prepare the 1:10 tube. Use the original, undiluted sample to prepare the 1:10 dilution)<br /> # Prepare a 1 mg/mL stock solution of BSA by dissolving BSA in ddH2O. Prepare a dilution series using ddH2O (or whatever buffer/media is appropriate) in 1.5 mL tubes: 1 mg/mL, 0.8 mg/mL, 0.6 mg/mL, 0.4 mg/mL, and 0.2 mg/mL.<br /> # See '''Figure 1''' for a general plate setup. Using the reverse pipetting technique, pipet 160 μL of blank, standard, and samples into each appropriate well. Avoid creating air bubbles.<br /> # Spin down the plate at 300 rpm for 1 min to remove bubbles and any liquid on the side of the well.<br /> # Dump 4 mL of filtered Bradford Dye into a multichannel pipette reservoir. Using a multichannel pipette, pipet 40 μL of dye into each well. If a multichannel pipette is not available, '''''quickly''''' pipet 40 μL of dye into each well using a P200 pipette. <br /> # Incubate for at least 5 minutes at room temperature. <br /> # Take off the lid of the plate, and place it into a plate reader/spectrometer.<br /> # Measure absorbance at 595 nm. Export results to Excel. <br /> [[File:Bradford plate layout.png|thumb|'''Figure 1.''' General plate layout |border|center]]<br /> <br /> <br /> <br /> ''Analysis using the standard curve''<br /> <br /> # Export results from spectrometer to Excel.<br /> # Subtract the average absorbance of each well by the average absorbance of the blank.<br /> # Plot the known standard concentrations (X-axis; 0.2, 0.4, 0.6, 0.8, and 1.0 mg/mL) with the average absorbance at each concentration (Y-axis). '''Add a line of best fit and display the equation with R&lt;sup&gt;2&lt;/sup&gt; value.''' Ideally, the R&lt;sup&gt;2&lt;/sup&gt; value should be ≥ 0.99. R&lt;sup&gt;2&lt;/sup&gt; &lt; 0.94 curves should not be used. <br /> # Calculate the average absorbance for the samples of interest by using the equation of the line graph y = mx+b (Absorbance = Slope*Concentration + b) and solve for x.<br /> # If the calculated concentration falls within the linear range of the standard curve (0.2 - 1.0 mg/mL), then it is accurate. If necessary, determine the concentration of the undiluted sample by multiplying by the dilution factor. <br /> <br /> # <br /> <br /> #</div>

Dicksonb https://wiki.phagocytes.ca/index.php?title=Bradford_Assay&diff=137 2022-01-15T14:20:54Z

<p>Dicksonb: </p> <hr /> <div>'''Introduction'''<br /> <br /> The Bradford assay should be used to quantify total protein within a sample relative to a standard curve. This is important when running proteins on SDS-PAGE/Western blot, where each lane must contain an identical amount of total protein. This way, the intensity of the bands can be compared between different lanes, as standardizing the amount of protein loaded per lane will account for any variation in cell count from which the proteins were derived. <br /> <br /> '''Protocol'''<br /> <br /> ''Some things to know'':<br /> <br /> * This protocol uses Bio-Rad products<br /> * This protocol assumes the use of bovine serum albumin (BSA) for the standard curve, which has a linear range of 200 μg/mL to 1000 μg/mL within Bio-Rad's protein assay dye. Linear range may be improved by using other products, but are more expensive than Bio-Rad's. <br /> * If your protein is within a supernatant containing an acid indicator, such as phenol red, this may interfere with the assay. If your protein is within a solution containing phenol red, then ensure that all samples, standards, and blanks are diluted with that media (e.g. Serum free DMEM).<br /> * This protocol assumes all standards, samples, and blanks are run in triplicates.<br /> * Each tube will have extra volume to ensure you have enough for the last replicate. <br /> * Reverse pipetting technique is highly recommended to avoid air bubbles and to ensure accuracy. <br /> * The Bradford Dye is a 5X stock solution.<br /> <br /> ''For a 96 well plate''<br /> <br /> # Using a 0.2 μm filter and 15 mL syringe, filter approximately 4 mL of Bio-Rad Protein Assay Dye Reagent Concentrate (Bradford Dye) into a 5 mL Eppendorf tube or 15 mL falcon tube. Let it warm to room temperature. <br /> # Collect lysate or supernatant that which the protein concentration needs to be calculated for. Keep on ice. <br /> # In separate 1.5 mL tubes, dilute the sample using ddH2O (or whatever buffer/media is appropriate) into 1:2 and 1:10 dilutions into a total of 600 μL. ('''NOTE:''' Do not use the 1:2 diluted sample to prepare the 1:10 tube. Use the original, undiluted sample to prepare the 1:10 dilution)<br /> # Prepare a 1 mg/mL stock solution of BSA by dissolving BSA in ddH2O. Prepare a dilution series using ddH2O (or whatever buffer/media is appropriate) in 1.5 mL tubes: 1 mg/mL, 0.8 mg/mL, 0.6 mg/mL, 0.4 mg/mL, and 0.2 mg/mL.<br /> # See '''Figure 1''' for a general plate setup. Using the reverse pipetting technique, pipet 160 μL of blank, standard, and samples into each appropriate well. Avoid creating air bubbles.<br /> # Spin down the plate at 300 rpm for 1 min to remove bubbles and any liquid on the side of the well.<br /> # Dump 4 mL of filtered Bradford Dye into a multichannel pipette reservoir. Using a multichannel pipette, pipet 40 μL of dye into each well. If a multichannel pipette is not available, '''''quickly''''' pipet 40 μL of dye into each well using a P200 pipette. <br /> # Incubate for at least 5 minutes at room temperature. <br /> # Take off the lid of the plate, and place it into a plate reader/spectrometer.<br /> # Measure absorbance at 595 nm. Export results to Excel. <br /> [[File:Bradford plate layout.png|left|thumb|'''Figure 1.''' General plate layout ]]<br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> ''Analysis using the standard curve''<br /> <br /> # Export results from spectrometer to Excel.<br /> # Subtract the average absorbance of each well by the average absorbance of the blank.<br /> # Plot the known standard concentrations (X-axis; 0.2, 0.4, 0.6, 0.8, and 1.0 mg/mL) with the average absorbance at each concentration (Y-axis). '''Add a line of best fit and display the equation with R&lt;sup&gt;2&lt;/sup&gt; value.''' Ideally, the R&lt;sup&gt;2&lt;/sup&gt; value should be ≥ 0.99. R&lt;sup&gt;2&lt;/sup&gt; &lt; 0.94 curves should not be used. <br /> # Calculate the average absorbance for the samples of interest by using the equation of the line graph y = mx+b (Absorbance = Slope*Concentration + b) and solve for x.<br /> # If the calculated concentration falls within the linear range of the standard curve (0.2 - 1.0 mg/mL), then it is accurate. If necessary, determine the concentration of the undiluted sample by multiplying by the dilution factor. <br /> <br /> # <br /> <br /> #</div>

Dicksonb https://wiki.phagocytes.ca/index.php?title=Bradford_Assay&diff=136 2022-01-14T18:47:17Z

<p>Dicksonb: </p> <hr /> <div>'''Introduction'''<br /> <br /> The Bradford assay should be used to quantify total protein within a sample relative to a standard curve. This is important when running proteins on SDS-PAGE/Western blot, where each lane must contain an identical amount of total protein. This way, the intensity of the bands can be compared between different lanes, as standardizing the amount of protein loaded per lane will account for any variation in cell count from which the proteins were derived. <br /> <br /> '''Protocol'''<br /> <br /> ''Some things to know'':<br /> <br /> * This protocol uses Bio-Rad products<br /> * This protocol assumes the use of bovine serum albumin (BSA) for the standard curve, which has a linear range of 200 μg/mL to 1000 μg/mL within Bio-Rad's protein assay dye. Linear range may be improved by using other products, but are more expensive than Bio-Rad's. <br /> * If your protein is within a supernatant containing an acid indicator, such as phenol red, this may interfere with the assay. If your protein is within a solution containing phenol red, then ensure that all samples, standards, and blanks are diluted with that media (e.g. Serum free DMEM).<br /> * This protocol assumes all standards, samples, and blanks are run in triplicates.<br /> * Each tube will have extra volume to ensure you have enough for the last replicate. <br /> * Reverse pipetting technique is highly recommended to avoid air bubbles and to ensure accuracy. <br /> * The Bradford Dye is a 5X stock solution.<br /> <br /> ''For a 96 well plate''<br /> <br /> # Using a 0.2 μm filter and 15 mL syringe, filter approximately 4 mL of Bio-Rad Protein Assay Dye Reagent Concentrate (Bradford Dye) into a 5 mL Eppendorf tube or 15 mL falcon tube. Let it warm to room temperature. <br /> # Collect lysate or supernatant that which the protein concentration needs to be calculated for. Keep on ice. <br /> # In separate 1.5 mL tubes, dilute the sample using ddH2O (or whatever buffer/media is appropriate) into 1:2 and 1:10 dilutions into a total of 600 μL. ('''NOTE:''' Do not use the 1:2 diluted sample to prepare the 1:10 tube. Use the original, undiluted sample to prepare the 1:10 dilution)<br /> # Using a 2 mg/mL BSA stock solution (Bio-Rad), prepare a dilution series using ddH2O (or whatever buffer/media is appropriate) in 1.5 mL tubes: 1 mg/mL, 0.8 mg/mL, 0.6 mg/mL, 0.4 mg/mL, and 0.2 mg/mL. See '''Figure''' '''1.''' <br /> # See '''Figure 2''' for a general plate setup. Using the reverse pipetting technique, pipet 160 μL of blank, standard, and samples into each appropriate well. Avoid creating air bubbles.<br /> # Spin down the plate at 300 rpm for 1 min to remove bubbles and any liquid on the side of the well.<br /> # Dump 4 mL of filtered Bradford Dye into a multichannel pipette reservoir. Using a multichannel pipette, pipet 40 μL of dye into each well. If a multichannel pipette is not available, '''''quickly''''' pipet 40 μL of dye into each well using a P200 pipette. <br /> # Incubate for at least 5 minutes at room temperature. <br /> # Take off the lid of the plate, and place it into a plate reader/spectrometer.<br /> # Measure absorbance at 595 nm. Export results to Excel. <br /> [[File:Bradford plate layout.png|left|thumb|'''Figure 1.''' General plate layout ]]<br /> <br /> ''Analysis using the standard curve''<br /> <br /> # <br /> <br /> #</div>

Dicksonb https://wiki.phagocytes.ca/index.php?title=File:Bradford_plate_layout.png&diff=135 2022-01-14T18:46:06Z

<p>Dicksonb: </p> <hr /> <div>General layout of a 96 well plate for the Bradford Assay. Figure by Brandon Dickson.</div>

Dicksonb https://wiki.phagocytes.ca/index.php?title=Bradford_Assay&diff=134 2022-01-14T03:00:34Z

<p>Dicksonb: </p> <hr /> <div>'''Introduction'''<br /> <br /> The Bradford assay should be used to quantify total protein within a sample relative to a standard curve. This is important when running proteins on SDS-PAGE/Western blot, where each lane must contain an identical amount of total protein. This way, the intensity of the bands can be compared between different lanes, as standardizing the amount of protein loaded per lane will account for any variation in cell count from which the proteins were derived. <br /> <br /> '''Protocol'''<br /> <br /> ''Some things to know'':<br /> <br /> * This protocol uses Bio-Rad products<br /> * This protocol assumes the use of bovine serum albumin (BSA) for the standard curve, which has a linear range of 200 μg/mL to 1000 μg/mL within Bio-Rad's protein assay dye. Linear range may be improved by using other products, but are more expensive than Bio-Rad's. <br /> * If your protein is within a supernatant containing an acid indicator, such as phenol red, this may interfere with the assay. If your protein is within a solution containing phenol red, then ensure that all samples, standards, and blanks are diluted with that media (e.g. Serum free DMEM).<br /> * This protocol assumes all standards, samples, and blanks are run in triplicates.<br /> * Each tube will have extra volume to ensure you have enough for the last replicate. <br /> * Reverse pipetting technique is highly recommended to avoid air bubbles and to ensure accuracy. <br /> * The Bradford Dye is a 5X stock solution.<br /> <br /> ''For a 96 well plate''<br /> <br /> # Using a 0.2 μm filter and 15 mL syringe, filter approximately 4 mL of Bio-Rad Protein Assay Dye Reagent Concentrate (Bradford Dye) into a 5 mL Eppendorf tube or 15 mL falcon tube. Let it warm to room temperature. <br /> # Collect lysate or supernatant that which the protein concentration needs to be calculated for. Keep on ice. <br /> # In separate 1.5 mL tubes, dilute the sample using ddH2O (or whatever buffer/media is appropriate) into 1:2 and 1:10 dilutions into a total of 600 μL. ('''NOTE:''' Do not use the 1:2 diluted sample to prepare the 1:10 tube. Use the original, undiluted sample to prepare the 1:10 dilution)<br /> # Using a 2 mg/mL BSA stock solution (Bio-Rad), prepare a dilution series using ddH2O (or whatever buffer/media is appropriate) in 1.5 mL tubes: 1 mg/mL, 0.8 mg/mL, 0.6 mg/mL, 0.4 mg/mL, and 0.2 mg/mL. See '''Figure''' '''1.''' <br /> # See '''Figure 2''' for a general plate setup. Using the reverse pipetting technique, pipet 160 μL of blank, standard, and samples into each appropriate well. Avoid creating air bubbles.<br /> # Spin down the plate at 300 rpm for 1 min to remove bubbles and any liquid on the side of the well.<br /> # Dump 4 mL of filtered Bradford Dye into a multichannel pipette reservoir. Using a multichannel pipette, pipet 40 μL of dye into each well. If a multichannel pipette is not available, '''''quickly''''' pipet 40 μL of dye into each well using a P200 pipette. <br /> # Incubate for at least 5 minutes at room temperature. <br /> # Take off the lid of the plate, and place it into a plate reader/spectrometer.<br /> # Measure absorbance at 595 nm. Export results to Excel. <br /> <br /> <br /> ''Analysis using the standard curve''<br /> <br /> # <br /> <br /> #</div>

Dicksonb https://wiki.phagocytes.ca/index.php?title=Main_Page&diff=133 2022-01-14T01:36:38Z

<p>Dicksonb: </p> <hr /> <div>[[File:PnW.png|thumb|300x300px|Visit our Lab's Webpage at [http://www.phagocytes.ca www.phagocytes.ca]]]<br /> Welcome to the protocol wiki for the lab of [http://phagocytes.ca Dr. Bryan Heit], at the [http://www.uwo.ca University of Western Ontario]. This site contains the protocols, and other information, we frequently use in our lab.&amp;nbsp; Only laboratory members may edit pages, but anyone is welcome to use these protocols.&amp;nbsp; If you use these protocol, please cite us.&amp;nbsp; A link for generating citations can be found on the left side of the page.<br /> <br /> &lt;br&gt; Consult the [http://meta.wikimedia.org/wiki/Help:Contents User's Guide] for information on using the wiki software.&lt;br&gt; &lt;br&gt;<br /> <br /> '''''Lab members:'''''<br /> <br /> *To get a wiki account please contact Dr. Heit. <br /> *Log in&amp;nbsp;(upper-right side of screen) to add/edit pages. &amp;nbsp; <br /> *Please follow [[Editing|these formatting instructions]] when editing/creating protocols. <br /> *To create a new page, [[Create new|follow these instructions]]. <br /> <br /> &lt;br&gt;<br /> <br /> = Index of Protocols =<br /> {| width=&quot;100%&quot;<br /> |- style=&quot;vertical-align:top;&quot;<br /> |<br /> ==== Lab Operations ====<br /> *[[Entrance Protocol]]<br /> *[[Exit Protocol]]<br /> *[[Labbook Guidelines|Guidelines for Proper Use of Laboratory Notebooks]]<br /> *[[Saving Experimental Files|Proper Saving of Experimental Files]]<br /> *[[Setting Up Network Drives|Setting up Access to Network Drives &amp;amp; Wiki]]<br /> |<br /> ==== DNA/Cloning ====<br /> <br /> *[[Colony PCR]]<br /> *[[PCR]]<br /> *[[Digests]]<br /> *[[Ligation]]<br /> *[[Gibson Assembly]]<br /> *[[Quick 'n' Easy Competent E. coli|Quick 'n' Easy Competent ''E. coli'' (Dh5a)]]<br /> *[[Competent e coli|Generating Competent ''E. coli'' (TFB, BL21 &amp; ZYCY10P3S2T)]]<br /> *[[E. coli Transduction|Transducing ''E. coli'']]<br /> *[[Transformation]]<br /> *[[Generating Minicircles]]<br /> |<br /> ==== Microscopy ====<br /> *[[Competition of Charged Molecules with Lipophilic Cations]]<br /> *[[Immunostaining|Fluorescent Immunostaining]]<br /> *[[Inhibition of Focal Contact Signaling]]<br /> *[[Single Particle Tracking]]<br /> *[[Staining for GSD]]<br /> *[[Reducing Photobleaching]]<br /> *[[Acid Washing Coverslips]]<br /> *[[Live Cell FRET]] (depreciated)<br /> *[[3D Printed PDMS Chambers]]<br /> *[[FRET in FIJI]]<br /> *[[Traction Force Microscopy]]<br /> |<br /> |- style=&quot;vertical-align:top;&quot;<br /> |<br /> ==== General Protocols ====<br /> *[[Common buffers|Common Buffers]]<br /> *[[Cell Culture Guidelines]]<br /> *[[Bacterial Growth Media]]<br /> *[[Primary Macrophage Culture]]<br /> *[[Freezing and Thawing Cells]]<br /> *[[Antibiotics]]<br /> *[[Antibiotic Plates]]<br /> *[[Agarose Gels]]<br /> *[[Water Bath Antibiotic Solution]]<br /> |<br /> ==== Protein work ====<br /> *[[Western Blotting]]<br /> *[[Immunoprecipitation]]<br /> *[[Fab preparation]]<br /> *[[Fab Purification by FPLC|Fab purification using FPLC]]<br /> *[[Updated FPLC Size Exclusion Procedure]]<br /> *[[Coomassie Staining]]<br /> *[[Receptor Cross-linking and Activation|Receptor activation by Cross-Linking]]<br /> *[[Stripping &amp; Reprobing Blots|Stripping &amp;amp; Reprobing Blots]]<br /> *[[Nitrogen Cavitation]]<br /> *[[Bradford Assay]]<br /> |<br /> ==== Lipids ====<br /> *[[Asymmetric liposomes]]<br /> *[[Lipid Coated Bead Preparation]]<br /> *[[Lipid Extraction from Cells]]<br /> *[[Lipisome and Lipid-Coated Beads]]<br /> |<br /> |- style=&quot;vertical-align:top;&quot;<br /> |<br /> ==== Phagocytosis Protocols ====<br /> <br /> *[[Opsonization]]<br /> *[[Preparation of Silica-Magnetic Beads]]<br /> *[[Synchronised Phagocytosis]]<br /> *[[Phagosome Isolation]]<br /> *[[Primary Human Macrophages]]<br /> *[[Labelled E coli]]<br /> *[[Inside-out Labelling of Bacteria]]<br /> *[[Gentamicin Protection Assay]]<br /> *[[Preparation of Digestion-Tracking Bacteria]]<br /> |<br /> ==== Cell Biology ====<br /> *[[Ablation of Recycling Endosomes]]<br /> *[[Cell-Type Specific Transfection Protocols]]<br /> *[[G418 &amp; Puromycin Kill Curves|G418 &amp;amp; Puromycin Kill Curves]]<br /> *[[Apoptosis Detection with AnnexinV and PI]]<br /> *[[Immuno-FISH]]<br /> |'''Transfections and Transductions'''<br /> * [[J774 Cell Transfection]]<br /> * [[Raw Cell Transfection]]<br /> * [[Neon® Transfection System]]<br /> * [[Titering Pseudo-typed Lentiviruses]]<br /> *[[Transduction of THP-1s]]<br /> <br /> *<br /> |}<br /> <br /> = Links to More Protocols=<br /> {| width=&quot;100%&quot;<br /> |- style=&quot;vertical-align:top;&quot;<br /> |<br /> ====General Protocol Sites====<br /> *[http://www.benchfly.com/ BenchFly] - Free Video Protocols<br /> *[http://www.protocol-online.org/ Protocols Online] - large database of biology protocols<br /> *[http://openwetware.org/wiki/Main_Page Open Wet Ware] - large database of open protocols<br /> *[http://www.molecularstation.com/protocol-links/ Molecular Station] - links to many lab-generated protocols<br /> *[http://www.thelabrat.com/protocols/ TheLabRat] - various protocols &amp;amp; lab resourses.<br /> *[http://www.thelabrat.com/protocols/reagents.shtml Common Buffer Recipes] @ TheLabRat<br /> *[http://www.rsc.org/Publishing/Journals/lc/Chips_and_Tips/index.asp Chips &amp;amp; Tips] - Microfluidics protocols<br /> |<br /> ====Free Science Ebooks====<br /> *[https://www.gitbook.com/book/petebankhead/imagej-intro/details Analyzing fluorescence microscopy images with ImageJ]<br /> *[http://www.imaging-git.com/applications/bioimage-data-analysis-0 Bioimage Data Analysis] - Free registration required<br /> <br /> |}<br /> <br /> =Useful Links=<br /> {| width=&quot;100%&quot;<br /> |- style=&quot;vertical-align:top;&quot;<br /> |<br /> ====Molecular Biology Databases====<br /> *[http://www.ncbi.nlm.nih.gov/gene Pubmed Gene] - Find gene information<br /> *[http://www.ncbi.nlm.nih.gov/refseq/rsg/ Pubmed RefSeq] - Find reference sequences<br /> *[http://www.uniprot.org/ Uniprot] - Find protein sequence &amp;amp; structure<br /> *[http://hapmap.ncbi.nlm.nih.gov/ HapMap] - Find human polymophisms<br /> *[http://www.ncbi.nlm.nih.gov/omim OMIM] - Find human gene-assocations<br /> *[http://www.pantherdb.org/ PANTHER] - Automated Protein Function Classification<br /> *[http://useast.ensembl.org/index.html BioGrid] - Protein Interactions<br /> *[http://useast.ensembl.org/index.html Ensemble Genome] - Browse multiple genomes<br /> *[http://www.proteinatlas.org/ Human Protein Atlas] - Info onn gene exrpession, antibody's, etc<br /> *[http://www.genecards.org/index.shtml Gene Cards] - Condenced information on genes<br /> *[http://www.ihop-net.org/UniPub/iHOP/ iHOP] - Information Hyperlinked Over Proteins<br /> *[http://www.hprd.org/index_html Human Protein Reference Database]<br /> *[http://www.wwpdb.org/ PDB] - Protein Structures<br /> *[http://genetics.bwh.harvard.edu/pph2/ PolyPhen2] - SNP Phenotype Predictor<br /> *[http://sift.jcvi.org/ SIFT] - SNP Phenotype Predictor/db<br /> *[http://www.timetree.org/index.php TimeTree] - evolutionary divergence database<br /> |<br /> ==== Molecular Biology Tools====<br /> *[http://blast.ncbi.nlm.nih.gov/Blast.cgi NCBI Blast]<br /> *[http://ca.expasy.org/ ExPASy Tools]<br /> *[http://www.basic.northwestern.edu/biotools/oligocalc.html OligoCalc] - PCR primer Tm calculator<br /> *[http://tools.neb.com/NEBcutter2/index.php NEB Cutter]<br /> *[http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/isoschizomers.asp NEB Isoschizomers]<br /> *[http://searchlauncher.bcm.tmc.edu/seq-util/Options/revcomp.html Reverse-Complement DNA Sequence]<br /> *[http://www.insilico.uni-duesseldorf.de/Lig_Input.html Ligation Calculator]<br /> *[http://www.addgene.org/ AddGene] - clone by e-mail!<br /> *[http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_pattinprot.html PattenProt] - search genomes for protein patterns<br /> *[http://workbench.sdsc.edu/ Biology Workbench]<br /> *[http://www.ebi.ac.uk/Tools/sequence.html EMBL Sequence Tools]<br /> *[http://www.ncbi.nlm.nih.gov/projects/gorf/ NCBI ORF Finder]<br /> *[http://searchlauncher.bcm.tmc.edu/ BCM Search Launcher]<br /> *[http://swissmodel.expasy.org/ SWISS Model protein modeling]<br /> *[http://searchlauncher.bcm.tmc.edu/seq-search/struc-predict.html Secondary Structure Prediction]<br /> *[http://www.predictprotein.org/ PredictProtein] - Protein structure prediction<br /> *[http://3d-alignment.eu/ STRAP] - Protein aligments with structure<br /> |<br /> =====Microscopy Tools===== <br /> *[http://fbs.robarts.ca/ London Regional Microscopy Facility Bookings]<br /> *[http://www.microscopyu.com/ Microscopy U] - Everything you want to know about microscopes<br /> *[http://jcb.rupress.org/content/166/1/11.full Paper on Image Processing Standards] - How not to loose your job<br /> *[http://www.invitrogen.com/site/us/en/home/support/Research-Tools/Fluorescence-SpectraViewer.html Fluorophore Spectra Viewer] at Life Technology<br /> *[http://www.mcb.arizona.edu/ipc/fret/ Fluorescent Spectra Database] - FRET and other<br /> *[http://www.mcb.arizona.edu/IPC/spectra_page.htm Yet More Spectra] - from Arizona University<br /> *[http://www.confocal-microscopy.org/ www.confocal-microscopy.org] - Data on LSM methods and equipment<br /> *[http://fiji.sc/wiki/index.php/Fiji FIJI] - &lt;u&gt;FREE&lt;/u&gt; imageJ based image processing program<br /> *[http://www.dspguide.com/pdfbook.htm Free] image processing textbook<br /> *[http://www.archive.org/details/Lectures_on_Image_Processing Lectures on image processing]<br /> *[http://vaa3d.org/ VAA3D] FREE viewer for large 3/4/5D datasets<br /> |<br /> |- style=&quot;vertical-align:top;&quot;<br /> |<br /> ====Journal Resources====<br /> *[http://www.ncbi.nlm.nih.gov/pubmed/ Pubmed]<br /> *[http://www.ncbi.nlm.nih.gov/pmc/ Pubmed Central (USA)]<br /> *[http://pubmedcentralcanada.ca/ Pubmed Central (Canada)]<br /> *[http://scholar.google.com Google Scholar]<br /> *[http://eigenfactor.org/ Eigenfactor] - free journal imapct scores<br /> |<br /> ====''In Vivo'' Tools====<br /> *[http://www.emouseatlas.org/emap/home.html EMAP] - Virtual mouse anatomy<br /> *[http://phenome.jax.org/ Mouse phenome database] - mouse phenotypes&lt;br&gt;<br /> |<br /> ====Protease Tools====<br /> *[http://merops.sanger.ac.uk/ MEROPS] - Peptidase database<br /> *[http://www.proteolysis.org/proteases PMAP] - Proteolysis Map<br /> *[http://casbase.org/casvm/index.html CASVM] - Caspace substrate prediction<br /> *[http://bioinf.gen.tcd.ie/casbah/ CASBAH] - Caspase cleaveage site database<br /> |<br /> |- style=&quot;vertical-align:top;&quot;<br /> |<br /> ====Genomics Resources====<br /> *[http://www.gwascentral.org GWAS Central]<br /> |<br /> ====Chemical Tools====<br /> <br /> *[http://www.chemspider.com/ ChemSpider] - General chemistry database<br /> *[http://pubchem.ncbi.nlm.nih.gov/ PubChem] - Chemical structure database<br /> |<br /> ====Lipid Tools====<br /> *[http://www.lipidmaps.org/ Lipid Maps] - Lipidomics gateway at ''Nature''<br /> *[http://www.avantilipids.com/ Avanti Lipids] - Buy lipids<br /> |}</div>

Dicksonb https://wiki.phagocytes.ca/index.php?title=Main_Page&diff=132 2022-01-14T01:35:45Z

<p>Dicksonb: </p> <hr /> <div>[[File:PnW.png|thumb|300x300px|Visit our Lab's Webpage at [http://www.phagocytes.ca www.phagocytes.ca]]]<br /> Welcome to the protocol wiki for the lab of [http://phagocytes.ca Dr. Bryan Heit], at the [http://www.uwo.ca University of Western Ontario]. This site contains the protocols, and other information, we frequently use in our lab.&amp;nbsp; Only laboratory members may edit pages, but anyone is welcome to use these protocols.&amp;nbsp; If you use these protocol, please cite us.&amp;nbsp; A link for generating citations can be found on the left side of the page.<br /> <br /> &lt;br&gt; Consult the [http://meta.wikimedia.org/wiki/Help:Contents User's Guide] for information on using the wiki software.&lt;br&gt; &lt;br&gt;<br /> <br /> '''''Lab members:'''''<br /> <br /> *To get a wiki account please contact Dr. Heit. <br /> *Log in&amp;nbsp;(upper-right side of screen) to add/edit pages. &amp;nbsp; <br /> *Please follow [[Editing|these formatting instructions]] when editing/creating protocols. <br /> *To create a new page, [[Create new|follow these instructions]]. <br /> <br /> &lt;br&gt;<br /> <br /> = Index of Protocols =<br /> {| width=&quot;100%&quot;<br /> |- style=&quot;vertical-align:top;&quot;<br /> |<br /> ==== Lab Operations ====<br /> *[[Entrance Protocol]]<br /> *[[Exit Protocol]]<br /> *[[Labbook Guidelines|Guidelines for Proper Use of Laboratory Notebooks]]<br /> *[[Saving Experimental Files|Proper Saving of Experimental Files]]<br /> *[[Setting Up Network Drives|Setting up Access to Network Drives &amp;amp; Wiki]]<br /> |<br /> ==== DNA/Cloning ====<br /> <br /> *[[Colony PCR]]<br /> *[[PCR]]<br /> *[[Digests]]<br /> *[[Ligation]]<br /> *[[Gibson Assembly]]<br /> *[[Quick 'n' Easy Competent E. coli|Quick 'n' Easy Competent ''E. coli'' (Dh5a)]]<br /> *[[Competent e coli|Generating Competent ''E. coli'' (TFB, BL21 &amp; ZYCY10P3S2T)]]<br /> *[[E. coli Transduction|Transducing ''E. coli'']]<br /> *[[Transformation]]<br /> *[[Generating Minicircles]]<br /> |<br /> ==== Microscopy ====<br /> *[[Competition of Charged Molecules with Lipophilic Cations]]<br /> *[[Immunostaining|Fluorescent Immunostaining]]<br /> *[[Inhibition of Focal Contact Signaling]]<br /> *[[Single Particle Tracking]]<br /> *[[Staining for GSD]]<br /> *[[Reducing Photobleaching]]<br /> *[[Acid Washing Coverslips]]<br /> *[[Live Cell FRET]] (depreciated)<br /> *[[3D Printed PDMS Chambers]]<br /> *[[FRET in FIJI]]<br /> *[[Traction Force Microscopy|Traction force microscopy]]<br /> |<br /> |- style=&quot;vertical-align:top;&quot;<br /> |<br /> ==== General Protocols ====<br /> *[[Common buffers|Common Buffers]]<br /> *[[Cell Culture Guidelines]]<br /> *[[Bacterial Growth Media]]<br /> *[[Primary Macrophage Culture]]<br /> *[[Freezing and Thawing Cells]]<br /> *[[Antibiotics]]<br /> *[[Antibiotic Plates]]<br /> *[[Agarose Gels]]<br /> *[[Water Bath Antibiotic Solution]]<br /> |<br /> ==== Protein work ====<br /> *[[Western Blotting]]<br /> *[[Immunoprecipitation]]<br /> *[[Fab preparation]]<br /> *[[Fab Purification by FPLC|Fab purification using FPLC]]<br /> *[[Updated FPLC Size Exclusion Procedure]]<br /> *[[Coomassie Staining]]<br /> *[[Receptor Cross-linking and Activation|Receptor activation by Cross-Linking]]<br /> *[[Stripping &amp; Reprobing Blots|Stripping &amp;amp; Reprobing Blots]]<br /> *[[Nitrogen Cavitation]]<br /> *[[Bradford Assay]]<br /> |<br /> ==== Lipids ====<br /> *[[Asymmetric liposomes]]<br /> *[[Lipid Coated Bead Preparation]]<br /> *[[Lipid Extraction from Cells]]<br /> *[[Lipisome and Lipid-Coated Beads]]<br /> |<br /> |- style=&quot;vertical-align:top;&quot;<br /> |<br /> ==== Phagocytosis Protocols ====<br /> <br /> *[[Opsonization]]<br /> *[[Preparation of Silica-Magnetic Beads]]<br /> *[[Synchronised Phagocytosis]]<br /> *[[Phagosome Isolation]]<br /> *[[Primary Human Macrophages]]<br /> *[[Labelled E coli]]<br /> *[[Inside-out Labelling of Bacteria]]<br /> *[[Gentamicin Protection Assay]]<br /> *[[Preparation of Digestion-Tracking Bacteria]]<br /> |<br /> ==== Cell Biology ====<br /> *[[Ablation of Recycling Endosomes]]<br /> *[[Cell-Type Specific Transfection Protocols]]<br /> *[[G418 &amp; Puromycin Kill Curves|G418 &amp;amp; Puromycin Kill Curves]]<br /> *[[Apoptosis Detection with AnnexinV and PI]]<br /> *[[Immuno-FISH]]<br /> |'''Transfections and Transductions'''<br /> * [[J774 Cell Transfection]]<br /> * [[Raw Cell Transfection]]<br /> * [[Neon® Transfection System]]<br /> * [[Titering Pseudo-typed Lentiviruses]]<br /> *[[Transduction of THP-1s]]<br /> <br /> *<br /> |}<br /> <br /> = Links to More Protocols=<br /> {| width=&quot;100%&quot;<br /> |- style=&quot;vertical-align:top;&quot;<br /> |<br /> ====General Protocol Sites====<br /> *[http://www.benchfly.com/ BenchFly] - Free Video Protocols<br /> *[http://www.protocol-online.org/ Protocols Online] - large database of biology protocols<br /> *[http://openwetware.org/wiki/Main_Page Open Wet Ware] - large database of open protocols<br /> *[http://www.molecularstation.com/protocol-links/ Molecular Station] - links to many lab-generated protocols<br /> *[http://www.thelabrat.com/protocols/ TheLabRat] - various protocols &amp;amp; lab resourses.<br /> *[http://www.thelabrat.com/protocols/reagents.shtml Common Buffer Recipes] @ TheLabRat<br /> *[http://www.rsc.org/Publishing/Journals/lc/Chips_and_Tips/index.asp Chips &amp;amp; Tips] - Microfluidics protocols<br /> |<br /> ====Free Science Ebooks====<br /> *[https://www.gitbook.com/book/petebankhead/imagej-intro/details Analyzing fluorescence microscopy images with ImageJ]<br /> *[http://www.imaging-git.com/applications/bioimage-data-analysis-0 Bioimage Data Analysis] - Free registration required<br /> <br /> |}<br /> <br /> =Useful Links=<br /> {| width=&quot;100%&quot;<br /> |- style=&quot;vertical-align:top;&quot;<br /> |<br /> ====Molecular Biology Databases====<br /> *[http://www.ncbi.nlm.nih.gov/gene Pubmed Gene] - Find gene information<br /> *[http://www.ncbi.nlm.nih.gov/refseq/rsg/ Pubmed RefSeq] - Find reference sequences<br /> *[http://www.uniprot.org/ Uniprot] - Find protein sequence &amp;amp; structure<br /> *[http://hapmap.ncbi.nlm.nih.gov/ HapMap] - Find human polymophisms<br /> *[http://www.ncbi.nlm.nih.gov/omim OMIM] - Find human gene-assocations<br /> *[http://www.pantherdb.org/ PANTHER] - Automated Protein Function Classification<br /> *[http://useast.ensembl.org/index.html BioGrid] - Protein Interactions<br /> *[http://useast.ensembl.org/index.html Ensemble Genome] - Browse multiple genomes<br /> *[http://www.proteinatlas.org/ Human Protein Atlas] - Info onn gene exrpession, antibody's, etc<br /> *[http://www.genecards.org/index.shtml Gene Cards] - Condenced information on genes<br /> *[http://www.ihop-net.org/UniPub/iHOP/ iHOP] - Information Hyperlinked Over Proteins<br /> *[http://www.hprd.org/index_html Human Protein Reference Database]<br /> *[http://www.wwpdb.org/ PDB] - Protein Structures<br /> *[http://genetics.bwh.harvard.edu/pph2/ PolyPhen2] - SNP Phenotype Predictor<br /> *[http://sift.jcvi.org/ SIFT] - SNP Phenotype Predictor/db<br /> *[http://www.timetree.org/index.php TimeTree] - evolutionary divergence database<br /> |<br /> ==== Molecular Biology Tools====<br /> *[http://blast.ncbi.nlm.nih.gov/Blast.cgi NCBI Blast]<br /> *[http://ca.expasy.org/ ExPASy Tools]<br /> *[http://www.basic.northwestern.edu/biotools/oligocalc.html OligoCalc] - PCR primer Tm calculator<br /> *[http://tools.neb.com/NEBcutter2/index.php NEB Cutter]<br /> *[http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/isoschizomers.asp NEB Isoschizomers]<br /> *[http://searchlauncher.bcm.tmc.edu/seq-util/Options/revcomp.html Reverse-Complement DNA Sequence]<br /> *[http://www.insilico.uni-duesseldorf.de/Lig_Input.html Ligation Calculator]<br /> *[http://www.addgene.org/ AddGene] - clone by e-mail!<br /> *[http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_pattinprot.html PattenProt] - search genomes for protein patterns<br /> *[http://workbench.sdsc.edu/ Biology Workbench]<br /> *[http://www.ebi.ac.uk/Tools/sequence.html EMBL Sequence Tools]<br /> *[http://www.ncbi.nlm.nih.gov/projects/gorf/ NCBI ORF Finder]<br /> *[http://searchlauncher.bcm.tmc.edu/ BCM Search Launcher]<br /> *[http://swissmodel.expasy.org/ SWISS Model protein modeling]<br /> *[http://searchlauncher.bcm.tmc.edu/seq-search/struc-predict.html Secondary Structure Prediction]<br /> *[http://www.predictprotein.org/ PredictProtein] - Protein structure prediction<br /> *[http://3d-alignment.eu/ STRAP] - Protein aligments with structure<br /> |<br /> =====Microscopy Tools===== <br /> *[http://fbs.robarts.ca/ London Regional Microscopy Facility Bookings]<br /> *[http://www.microscopyu.com/ Microscopy U] - Everything you want to know about microscopes<br /> *[http://jcb.rupress.org/content/166/1/11.full Paper on Image Processing Standards] - How not to loose your job<br /> *[http://www.invitrogen.com/site/us/en/home/support/Research-Tools/Fluorescence-SpectraViewer.html Fluorophore Spectra Viewer] at Life Technology<br /> *[http://www.mcb.arizona.edu/ipc/fret/ Fluorescent Spectra Database] - FRET and other<br /> *[http://www.mcb.arizona.edu/IPC/spectra_page.htm Yet More Spectra] - from Arizona University<br /> *[http://www.confocal-microscopy.org/ www.confocal-microscopy.org] - Data on LSM methods and equipment<br /> *[http://fiji.sc/wiki/index.php/Fiji FIJI] - &lt;u&gt;FREE&lt;/u&gt; imageJ based image processing program<br /> *[http://www.dspguide.com/pdfbook.htm Free] image processing textbook<br /> *[http://www.archive.org/details/Lectures_on_Image_Processing Lectures on image processing]<br /> *[http://vaa3d.org/ VAA3D] FREE viewer for large 3/4/5D datasets<br /> |<br /> |- style=&quot;vertical-align:top;&quot;<br /> |<br /> ====Journal Resources====<br /> *[http://www.ncbi.nlm.nih.gov/pubmed/ Pubmed]<br /> *[http://www.ncbi.nlm.nih.gov/pmc/ Pubmed Central (USA)]<br /> *[http://pubmedcentralcanada.ca/ Pubmed Central (Canada)]<br /> *[http://scholar.google.com Google Scholar]<br /> *[http://eigenfactor.org/ Eigenfactor] - free journal imapct scores<br /> |<br /> ====''In Vivo'' Tools====<br /> *[http://www.emouseatlas.org/emap/home.html EMAP] - Virtual mouse anatomy<br /> *[http://phenome.jax.org/ Mouse phenome database] - mouse phenotypes&lt;br&gt;<br /> |<br /> ====Protease Tools====<br /> *[http://merops.sanger.ac.uk/ MEROPS] - Peptidase database<br /> *[http://www.proteolysis.org/proteases PMAP] - Proteolysis Map<br /> *[http://casbase.org/casvm/index.html CASVM] - Caspace substrate prediction<br /> *[http://bioinf.gen.tcd.ie/casbah/ CASBAH] - Caspase cleaveage site database<br /> |<br /> |- style=&quot;vertical-align:top;&quot;<br /> |<br /> ====Genomics Resources====<br /> *[http://www.gwascentral.org GWAS Central]<br /> |<br /> ====Chemical Tools====<br /> <br /> *[http://www.chemspider.com/ ChemSpider] - General chemistry database<br /> *[http://pubchem.ncbi.nlm.nih.gov/ PubChem] - Chemical structure database<br /> |<br /> ====Lipid Tools====<br /> *[http://www.lipidmaps.org/ Lipid Maps] - Lipidomics gateway at ''Nature''<br /> *[http://www.avantilipids.com/ Avanti Lipids] - Buy lipids<br /> |}</div>

Dicksonb https://wiki.phagocytes.ca/index.php?title=Traction_Force_Microscopy&diff=131 2022-01-14T01:34:43Z

<p>Dicksonb: In progress</p> <hr /> <div>'''Introduction'''<br /> <br /> Traction force microscopy (TFM) is used to measure the mechanical forces imposed by a cell on a thin linear elastic material (e.g. polyacrylamide) that is opsonized with a substrate of interest. For example, the polyacrylamide substrate can be opsonized by complement proteins, which are recognized by complement receptors on phagocytes to induce frustrated phagocytosis. The polyacrylamide substrate is embedded with fluorescent beads which serve as fiducial markers. TFM requires images of the &quot;unstrained&quot; state (the original position of the beads) and the &quot;strained state&quot; (the final position of the beads). 3rd party algorithms are used to measure the displacement of each fluorescent bead. Finally, algorithms are used to predict the force vectors (both magnitude and direction) required to cause these displacements.<br /> <br /> <br /> '''Protocol'''<br /> <br /> <br /> <br /> '''References'''<br /> <br /> &lt;ref&gt;Jaumouillé, V., Cartagena-Rivera, A. X. &amp; Waterman, C. M. Coupling of β2 integrins to actin by a mechanosensitive molecular clutch drives complement receptor-mediated phagocytosis. ''Nat Cell Biol'' '''21''', 1357–1369 (2019).<br /> <br /> &lt;/ref&gt;Jaumouillé, V., Cartagena-Rivera, A. X. &amp; Waterman, C. M. Coupling of β2 integrins to actin by a mechanosensitive molecular clutch drives complement receptor-mediated phagocytosis. Nat Cell Biol 21, 1357–1369 (2019).</div>

Dicksonb https://wiki.phagocytes.ca/index.php?title=Main_Page&diff=130 2022-01-13T15:59:25Z

<p>Dicksonb: </p> <hr /> <div>[[File:PnW.png|thumb|300x300px|Visit our Lab's Webpage at [http://www.phagocytes.ca www.phagocytes.ca]]]<br /> Welcome to the protocol wiki for the lab of [http://phagocytes.ca Dr. Bryan Heit], at the [http://www.uwo.ca University of Western Ontario]. This site contains the protocols, and other information, we frequently use in our lab.&amp;nbsp; Only laboratory members may edit pages, but anyone is welcome to use these protocols.&amp;nbsp; If you use these protocol, please cite us.&amp;nbsp; A link for generating citations can be found on the left side of the page.<br /> <br /> &lt;br&gt; Consult the [http://meta.wikimedia.org/wiki/Help:Contents User's Guide] for information on using the wiki software.&lt;br&gt; &lt;br&gt;<br /> <br /> '''''Lab members:'''''<br /> <br /> *To get a wiki account please contact Dr. Heit. <br /> *Log in&amp;nbsp;(upper-right side of screen) to add/edit pages. &amp;nbsp; <br /> *Please follow [[Editing|these formatting instructions]] when editing/creating protocols. <br /> *To create a new page, [[Create new|follow these instructions]]. <br /> <br /> &lt;br&gt;<br /> <br /> = Index of Protocols =<br /> {| width=&quot;100%&quot;<br /> |- style=&quot;vertical-align:top;&quot;<br /> |<br /> ==== Lab Operations ====<br /> *[[Entrance Protocol]]<br /> *[[Exit Protocol]]<br /> *[[Labbook Guidelines|Guidelines for Proper Use of Laboratory Notebooks]]<br /> *[[Saving Experimental Files|Proper Saving of Experimental Files]]<br /> *[[Setting Up Network Drives|Setting up Access to Network Drives &amp;amp; Wiki]]<br /> |<br /> ==== DNA/Cloning ====<br /> <br /> *[[Colony PCR]]<br /> *[[PCR]]<br /> *[[Digests]]<br /> *[[Ligation]]<br /> *[[Gibson Assembly]]<br /> *[[Quick 'n' Easy Competent E. coli|Quick 'n' Easy Competent ''E. coli'' (Dh5a)]]<br /> *[[Competent e coli|Generating Competent ''E. coli'' (TFB, BL21 &amp; ZYCY10P3S2T)]]<br /> *[[E. coli Transduction|Transducing ''E. coli'']]<br /> *[[Transformation]]<br /> *[[Generating Minicircles]]<br /> |<br /> ==== Microscopy ====<br /> *[[Competition of Charged Molecules with Lipophilic Cations]]<br /> *[[Immunostaining|Fluorescent Immunostaining]]<br /> *[[Inhibition of Focal Contact Signaling]]<br /> *[[Single Particle Tracking]]<br /> *[[Staining for GSD]]<br /> *[[Reducing Photobleaching]]<br /> *[[Acid Washing Coverslips]]<br /> *[[Live Cell FRET]] (depreciated)<br /> *[[3D Printed PDMS Chambers]]<br /> *[[FRET in FIJI]]<br /> |<br /> |- style=&quot;vertical-align:top;&quot;<br /> |<br /> ==== General Protocols ====<br /> *[[Common buffers|Common Buffers]]<br /> *[[Cell Culture Guidelines]]<br /> *[[Bacterial Growth Media]]<br /> *[[Primary Macrophage Culture]]<br /> *[[Freezing and Thawing Cells]]<br /> *[[Antibiotics]]<br /> *[[Antibiotic Plates]]<br /> *[[Agarose Gels]]<br /> *[[Water Bath Antibiotic Solution]]<br /> |<br /> ==== Protein work ====<br /> *[[Western Blotting]]<br /> *[[Immunoprecipitation]]<br /> *[[Fab preparation]]<br /> *[[Fab Purification by FPLC|Fab purification using FPLC]]<br /> *[[Updated FPLC Size Exclusion Procedure]]<br /> *[[Coomassie Staining]]<br /> *[[Receptor Cross-linking and Activation|Receptor activation by Cross-Linking]]<br /> *[[Stripping &amp; Reprobing Blots|Stripping &amp;amp; Reprobing Blots]]<br /> *[[Nitrogen Cavitation]]<br /> *[[Bradford Assay]]<br /> |<br /> ==== Lipids ====<br /> *[[Asymmetric liposomes]]<br /> *[[Lipid Coated Bead Preparation]]<br /> *[[Lipid Extraction from Cells]]<br /> *[[Lipisome and Lipid-Coated Beads]]<br /> |<br /> |- style=&quot;vertical-align:top;&quot;<br /> |<br /> ==== Phagocytosis Protocols ====<br /> <br /> *[[Opsonization]]<br /> *[[Preparation of Silica-Magnetic Beads]]<br /> *[[Synchronised Phagocytosis]]<br /> *[[Phagosome Isolation]]<br /> *[[Primary Human Macrophages]]<br /> *[[Labelled E coli]]<br /> *[[Inside-out Labelling of Bacteria]]<br /> *[[Gentamicin Protection Assay]]<br /> *[[Preparation of Digestion-Tracking Bacteria]]<br /> |<br /> ==== Cell Biology ====<br /> *[[Ablation of Recycling Endosomes]]<br /> *[[Cell-Type Specific Transfection Protocols]]<br /> *[[G418 &amp; Puromycin Kill Curves|G418 &amp;amp; Puromycin Kill Curves]]<br /> *[[Apoptosis Detection with AnnexinV and PI]]<br /> *[[Immuno-FISH]]<br /> |'''Transfections and Transductions'''<br /> * [[J774 Cell Transfection]]<br /> * [[Raw Cell Transfection]]<br /> * [[Neon® Transfection System]]<br /> * [[Titering Pseudo-typed Lentiviruses]]<br /> *[[Transduction of THP-1s]]<br /> <br /> *<br /> |}<br /> <br /> = Links to More Protocols=<br /> {| width=&quot;100%&quot;<br /> |- style=&quot;vertical-align:top;&quot;<br /> |<br /> ====General Protocol Sites====<br /> *[http://www.benchfly.com/ BenchFly] - Free Video Protocols<br /> *[http://www.protocol-online.org/ Protocols Online] - large database of biology protocols<br /> *[http://openwetware.org/wiki/Main_Page Open Wet Ware] - large database of open protocols<br /> *[http://www.molecularstation.com/protocol-links/ Molecular Station] - links to many lab-generated protocols<br /> *[http://www.thelabrat.com/protocols/ TheLabRat] - various protocols &amp;amp; lab resourses.<br /> *[http://www.thelabrat.com/protocols/reagents.shtml Common Buffer Recipes] @ TheLabRat<br /> *[http://www.rsc.org/Publishing/Journals/lc/Chips_and_Tips/index.asp Chips &amp;amp; Tips] - Microfluidics protocols<br /> |<br /> ====Free Science Ebooks====<br /> *[https://www.gitbook.com/book/petebankhead/imagej-intro/details Analyzing fluorescence microscopy images with ImageJ]<br /> *[http://www.imaging-git.com/applications/bioimage-data-analysis-0 Bioimage Data Analysis] - Free registration required<br /> <br /> |}<br /> <br /> =Useful Links=<br /> {| width=&quot;100%&quot;<br /> |- style=&quot;vertical-align:top;&quot;<br /> |<br /> ====Molecular Biology Databases====<br /> *[http://www.ncbi.nlm.nih.gov/gene Pubmed Gene] - Find gene information<br /> *[http://www.ncbi.nlm.nih.gov/refseq/rsg/ Pubmed RefSeq] - Find reference sequences<br /> *[http://www.uniprot.org/ Uniprot] - Find protein sequence &amp;amp; structure<br /> *[http://hapmap.ncbi.nlm.nih.gov/ HapMap] - Find human polymophisms<br /> *[http://www.ncbi.nlm.nih.gov/omim OMIM] - Find human gene-assocations<br /> *[http://www.pantherdb.org/ PANTHER] - Automated Protein Function Classification<br /> *[http://useast.ensembl.org/index.html BioGrid] - Protein Interactions<br /> *[http://useast.ensembl.org/index.html Ensemble Genome] - Browse multiple genomes<br /> *[http://www.proteinatlas.org/ Human Protein Atlas] - Info onn gene exrpession, antibody's, etc<br /> *[http://www.genecards.org/index.shtml Gene Cards] - Condenced information on genes<br /> *[http://www.ihop-net.org/UniPub/iHOP/ iHOP] - Information Hyperlinked Over Proteins<br /> *[http://www.hprd.org/index_html Human Protein Reference Database]<br /> *[http://www.wwpdb.org/ PDB] - Protein Structures<br /> *[http://genetics.bwh.harvard.edu/pph2/ PolyPhen2] - SNP Phenotype Predictor<br /> *[http://sift.jcvi.org/ SIFT] - SNP Phenotype Predictor/db<br /> *[http://www.timetree.org/index.php TimeTree] - evolutionary divergence database<br /> |<br /> ==== Molecular Biology Tools====<br /> *[http://blast.ncbi.nlm.nih.gov/Blast.cgi NCBI Blast]<br /> *[http://ca.expasy.org/ ExPASy Tools]<br /> *[http://www.basic.northwestern.edu/biotools/oligocalc.html OligoCalc] - PCR primer Tm calculator<br /> *[http://tools.neb.com/NEBcutter2/index.php NEB Cutter]<br /> *[http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/isoschizomers.asp NEB Isoschizomers]<br /> *[http://searchlauncher.bcm.tmc.edu/seq-util/Options/revcomp.html Reverse-Complement DNA Sequence]<br /> *[http://www.insilico.uni-duesseldorf.de/Lig_Input.html Ligation Calculator]<br /> *[http://www.addgene.org/ AddGene] - clone by e-mail!<br /> *[http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_pattinprot.html PattenProt] - search genomes for protein patterns<br /> *[http://workbench.sdsc.edu/ Biology Workbench]<br /> *[http://www.ebi.ac.uk/Tools/sequence.html EMBL Sequence Tools]<br /> *[http://www.ncbi.nlm.nih.gov/projects/gorf/ NCBI ORF Finder]<br /> *[http://searchlauncher.bcm.tmc.edu/ BCM Search Launcher]<br /> *[http://swissmodel.expasy.org/ SWISS Model protein modeling]<br /> *[http://searchlauncher.bcm.tmc.edu/seq-search/struc-predict.html Secondary Structure Prediction]<br /> *[http://www.predictprotein.org/ PredictProtein] - Protein structure prediction<br /> *[http://3d-alignment.eu/ STRAP] - Protein aligments with structure<br /> |<br /> =====Microscopy Tools===== <br /> *[http://fbs.robarts.ca/ London Regional Microscopy Facility Bookings]<br /> *[http://www.microscopyu.com/ Microscopy U] - Everything you want to know about microscopes<br /> *[http://jcb.rupress.org/content/166/1/11.full Paper on Image Processing Standards] - How not to loose your job<br /> *[http://www.invitrogen.com/site/us/en/home/support/Research-Tools/Fluorescence-SpectraViewer.html Fluorophore Spectra Viewer] at Life Technology<br /> *[http://www.mcb.arizona.edu/ipc/fret/ Fluorescent Spectra Database] - FRET and other<br /> *[http://www.mcb.arizona.edu/IPC/spectra_page.htm Yet More Spectra] - from Arizona University<br /> *[http://www.confocal-microscopy.org/ www.confocal-microscopy.org] - Data on LSM methods and equipment<br /> *[http://fiji.sc/wiki/index.php/Fiji FIJI] - &lt;u&gt;FREE&lt;/u&gt; imageJ based image processing program<br /> *[http://www.dspguide.com/pdfbook.htm Free] image processing textbook<br /> *[http://www.archive.org/details/Lectures_on_Image_Processing Lectures on image processing]<br /> *[http://vaa3d.org/ VAA3D] FREE viewer for large 3/4/5D datasets<br /> |<br /> |- style=&quot;vertical-align:top;&quot;<br /> |<br /> ====Journal Resources====<br /> *[http://www.ncbi.nlm.nih.gov/pubmed/ Pubmed]<br /> *[http://www.ncbi.nlm.nih.gov/pmc/ Pubmed Central (USA)]<br /> *[http://pubmedcentralcanada.ca/ Pubmed Central (Canada)]<br /> *[http://scholar.google.com Google Scholar]<br /> *[http://eigenfactor.org/ Eigenfactor] - free journal imapct scores<br /> |<br /> ====''In Vivo'' Tools====<br /> *[http://www.emouseatlas.org/emap/home.html EMAP] - Virtual mouse anatomy<br /> *[http://phenome.jax.org/ Mouse phenome database] - mouse phenotypes&lt;br&gt;<br /> |<br /> ====Protease Tools====<br /> *[http://merops.sanger.ac.uk/ MEROPS] - Peptidase database<br /> *[http://www.proteolysis.org/proteases PMAP] - Proteolysis Map<br /> *[http://casbase.org/casvm/index.html CASVM] - Caspace substrate prediction<br /> *[http://bioinf.gen.tcd.ie/casbah/ CASBAH] - Caspase cleaveage site database<br /> |<br /> |- style=&quot;vertical-align:top;&quot;<br /> |<br /> ====Genomics Resources====<br /> *[http://www.gwascentral.org GWAS Central]<br /> |<br /> ====Chemical Tools====<br /> <br /> *[http://www.chemspider.com/ ChemSpider] - General chemistry database<br /> *[http://pubchem.ncbi.nlm.nih.gov/ PubChem] - Chemical structure database<br /> |<br /> ====Lipid Tools====<br /> *[http://www.lipidmaps.org/ Lipid Maps] - Lipidomics gateway at ''Nature''<br /> *[http://www.avantilipids.com/ Avanti Lipids] - Buy lipids<br /> |}</div>

Dicksonb https://wiki.phagocytes.ca/index.php?title=Bradford_Assay&diff=129 2022-01-13T15:57:12Z

<p>Dicksonb: Started a new page for Bradford Assay - will finish when optimized</p> <hr /> <div>'''Introduction'''<br /> <br /> The Bradford assay should be used to quantify total protein within a sample relative to a standard curve. This is important when running proteins on SDS-PAGE/Western blot, where each lane must contain an identical amount of total protein. This way, the intensity of the bands can be compared between different lanes, as standardizing the amount of protein loaded per lane will account for any variation in cell count from which the proteins were derived. <br /> <br /> '''Protocol'''<br /> <br /> #</div>

Dicksonb https://wiki.phagocytes.ca/index.php?title=Bradford_Assay&diff=128 2022-01-13T15:51:53Z

<p>Dicksonb: The Bradford assay is used to determine the concentration of total protein within a sample relative to a standard curve.</p> <hr /> <div>Bradford Assay</div>

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