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| [[File:PnW.png|thumb|300x300px|Visit our Lab's Webpage at [http://www.phagocytes.ca www.phagocytes.ca]]]
| | This procedure was optimized using the 4G10 (anti-phosphotyrosine) antibody, but should work well for many other antibodies. For a generic protocol, add an additional 20-60min at RT fixing step after the 4C fixing step. New fixative is not needed, simply move the plate to the bench & cover with foil. Antibody concentrations will need to be optimized for each antibody. Please enter details for all optimized antibodies in the table at the end of the protocol.<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. Only laboratory members may edit pages, but anyone is welcome to use these protocols. If you use these protocol, please cite us. A link for generating citations can be found on the left side of the page.
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| <br> Consult the [http://meta.wikimedia.org/wiki/Help:Contents User's Guide] for information on using the wiki software.<br> <br>
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| '''''Lab members:'''''
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| *To get a wiki account please contact Dr. Heit.
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| *Log in (upper-right side of screen) to add/edit pages.
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| *Please follow [[Editing|these formatting instructions]] when editing/creating protocols.
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| *To create a new page, [[Create new|follow these instructions]].
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| <br>
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| = Index of Protocols =
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| {| width="100%" cellspacing="1" cellpadding="1" border="0" align="left"
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| ===== Lab Operations =====
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| *[[Entrance Protocol|Entrance Protocol]]
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| *[[Exit Protocol|Exit Protocol]]
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| *[[Labbook Guidelines|Guidelines for Proper Use of Laboratory Notebooks]]
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| *[[Saving Experimental Files|Proper Saving of Experimental Files]]
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| *[[Setting Up Network Drives|Setting up Access to Network Drives & Wiki]]
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| <br>
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| ===== General Protocols =====
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| *[[Common buffers|Common Buffers]]
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| *[[Cell Culture Guidelines|Cell Culture Guidelines]]
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| *[[Bacterial Growth Media|Bacterial Growth Media]]
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| *[[Primary Macrophage Culture|Primary Macrophage Culture]]
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| *[[Freezing and Thawing Cells|Freezing and Thawing Cells]]
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| *[[Raw Cell Transfection|Raw Cell Transfection]]
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| *[[J774 Cell Transfection|J774 Cell Transfection]]
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| *[[Antibiotics|Antibiotics]]
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| *[[Antibiotic Plates|Antibiotic Plates]]
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| *[[Agarose Gels|Agarose Gels]]
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| *[[Water Bath Antibiotic Solution|Water Bath Antibiotic Solution]]
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| <br>
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| ===== Phagocytosis Protocols =====
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| *[[Opsonization|Opsonization]]
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| *[[Preparation of Silica-Magnetic Beads|Preparation of Silica-Magnetic Beads]]
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| *[[Synchronised Phagocytosis|Synchronised Phagocytosis]]
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| *[[Phagosome Isolation|Phagosome Isolation]]
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| *[[Primary Human Macrophages|Primary Human Macrophages]]
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| *[[Labelled E coli|Labelled E coli]]
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| *[[Inside-out Labelling of Bacteria|Inside-out Labelling of Bacteria]]
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| *[[Gentamicin Protection Assay|Gentamicin Protection Assay]]
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| <br> <br>
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| ===== DNA/Cloning =====
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| *[[Colony PCR|Colony PCR]]<br>
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| *[[PCR|PCR]]
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| *[[Digests|Digests]]
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| *[[Ligation|Ligation]]
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| *[[Gibson Assembly|Gibson Assembly]]
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| *[[Quick 'n' Easy Competent E. coli|Quick 'n' Easy Competent ''E. coli'' (Dh5a)]]
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| *[[Competent e coli|Generating Competent ''E. coli'' (TFB, BL21 & ZYCY10P3S2T)]]
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| *[[E. coli Transduction|Transducing ''E. coli'']]
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| *[[Transformation|Transformation]]
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| *[[Neon® Transfection System|Neon® Transfection System]]
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| *[[Generating Minicircles|Generating Minicircles]]
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| <br>
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| ===== Protein work =====
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| *[[Western Blotting|Western Blotting]]
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| *[[Immunoprecipitation|Immunoprecipitation]]
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| *[[Fab preparation|Fab preparation]]
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| *[[Fab Purification by FPLC|Fab purification using FPLC]]
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| *[[Updated FPLC Size Exclusion Procedure|Updated FPLC Size Exclusion Procedure]]
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| *[[Coomassie Staining|Coomassie Staining]]
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| *[[Receptor Cross-linking and Activation|Receptor activation by Cross-Linking]]
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| *[[Stripping & Reprobing Blots|Stripping & Reprobing Blots]]
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| <br>
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| ===== Cell Biology<br> =====
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| *[[Ablation of Recycling Endosomes|Ablation of Recycling Endosomes]]
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| *[[Cell-Type Specific Transfection Protocols|Cell-Type Specific Transfection Protocols]]
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| *[[G418 & Puromycin Kill Curves|G418 & Puromycin Kill Curves]]
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| *[[Apoptosis Detection with AnnexinV and PI|Apoptosis Detection with AnnexinV and PI]]
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| ===== Microscopy =====
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| *[[Competition of Charged Molecules with Lipophilic Cations|Competition of Charged Molecules with Lipophilic Cations]]
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| *[[Immunostaining|Fluorescent Immunostaining]]
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| *[[Inhibition of Focal Contact Signaling|Inhibition of Focal Contact Signaling]]
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| *[[Single Particle Tracking|Single Particle Tracking]]
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| *[[Staining for GSD|Staining for GSD]]
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| *[[Reducing Photobleaching|Reducing Photobleaching]]
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| *[[Acid Washing Coverslips|Acid Washing Coverslips]]
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| *[[Live Cell FRET|Live Cell FRET]]
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| *[[3D Printed PDMS Chambers|3D Printed PDMS Chambers]]
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| <br>
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| ===== Lipids<br> =====
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| *[[Asymmetric liposomes|Asymmetric liposomes]]<br>
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| *[[Lipid Coated Bead Preparation|Lipid Coated Bead Preparation]]<br>
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| *[[Lipid Extraction from Cells|Lipid Extraction from Cells]]<br>
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| *[[Lipisome and Lipid-Coated Beads|Lipisome and Lipid-Coated Beads]]<br>
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| <br>
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| <br>
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| |}
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| = Links to More Protocols =
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| {| width="100%" cellspacing="1" cellpadding="1" border="0" align="left"
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| ===== General Protocol Sites =====
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| *[http://www.benchfly.com/ BenchFly] - Free Video Protocols
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| *[http://www.protocol-online.org/ Protocols Online] - large database of biology protocols
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| *[http://openwetware.org/wiki/Main_Page Open Wet Ware] - large database of open protocols
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| *[http://www.molecularstation.com/protocol-links/ Molecular Station] - links to many lab-generated protocols
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| *[http://www.thelabrat.com/protocols/ TheLabRat] - various protocols & lab resourses.
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| *[http://www.thelabrat.com/protocols/reagents.shtml Common Buffer Recipes] @ TheLabRat
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| *[http://www.rsc.org/Publishing/Journals/lc/Chips_and_Tips/index.asp Chips & Tips] - Microfluidics protocols
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| ===== Free Science Ebooks =====
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| *[https://www.gitbook.com/book/petebankhead/imagej-intro/details Analyzing fluorescence microscopy images with ImageJ]
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| *[http://www.imaging-git.com/applications/bioimage-data-analysis-0 Bioimage Data Analysis] - Free registration required
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| |}
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| = Useful Links =
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| {| width="100%" cellspacing="1" cellpadding="1" border="0"
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| ===== Molecular Biology Databases =====
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| *[http://www.ncbi.nlm.nih.gov/gene Pubmed Gene] - Find gene information<br>
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| *[http://www.ncbi.nlm.nih.gov/refseq/rsg/ Pubmed RefSeq] - Find reference sequences<br>
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| *[http://www.uniprot.org/ Uniprot] - Find protein sequence & structure<br>
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| *[http://hapmap.ncbi.nlm.nih.gov/ HapMap] - Find human polymophisms<br>
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| *[http://www.ncbi.nlm.nih.gov/omim OMIM] - Find human gene-assocations<br>
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| *[http://www.pantherdb.org/ PANTHER] - Automated Protein Function Classification
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| *[http://useast.ensembl.org/index.html BioGrid] - Protein Interactions<br>
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| *[http://useast.ensembl.org/index.html Ensemble Genome] - Browse multiple genomes<br>
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| *[http://www.proteinatlas.org/ Human Protein Atlas] - Info onn gene exrpession, antibody's, etc<br>
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| *[http://www.genecards.org/index.shtml Gene Cards] - Condenced information on genes<br>
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| *[http://www.ihop-net.org/UniPub/iHOP/ iHOP] - Information Hyperlinked Over Proteins<br>
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| *[http://www.hprd.org/index_html Human Protein Reference Database]<br>
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| *[http://www.wwpdb.org/ PDB] - Protein Structures<br>
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| *[http://genetics.bwh.harvard.edu/pph2/ PolyPhen2] - SNP Phenotype Predictor
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| *[http://sift.jcvi.org/ SIFT] - SNP Phenotype Predictor/db
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| *[http://www.timetree.org/index.php TimeTree] - evolutionary divergence database<br>
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| <br>
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| ===== Journal Resources<br> =====
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| *[http://www.ncbi.nlm.nih.gov/pubmed/ Pubmed]
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| *[http://www.ncbi.nlm.nih.gov/pmc/ Pubmed Central (USA)]
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| *[http://pubmedcentralcanada.ca/ Pubmed Central (Canada)]
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| *[http://scholar.google.com Google Scholar]
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| *[http://eigenfactor.org/ Eigenfactor] - free journal imapct scores
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| <br> | | <br> |
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| ===== Genomics Resources<br> ===== | | == Protocol == |
| *[http://www.gwascentral.org GWAS Central]
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| | #Fix cells in 4% PFA/PBS for 20' at room temperature. [https://www.ncbi.nlm.nih.gov/labs/pmc/articles/PMC4958280/ To preserve membrane structures], fix with 4% PFA in PEM, 37C, 10 min. |
| ===== Molecular Biology Tools =====
| | #Wash 3X PBS |
| | #Permeabilize and block using antibody buffer, 1hr at RT. |
| | #Add primary antibody at desired concentration in antibody buffer, 20C for 1 hour or 4C overnight. |
| | #*Use hanging drop method: |
| | #**Put 100ul antibody mixture on a piece of parafilm |
| | #**Flip coverslip onto drop |
| | #**Cover with foil or box and leave undisturbed for 20 min (surface staining) or up to overnight (intracellular staining; minimum 1 hr). '''KEEP LEVEL'''. |
| | #Was 3X 15min with PBS. |
| | #Add secondary antibody at desired concentration (typically 1:500 to 1:1000) in antibody buffer: |
| | #*Use hanging drop method: |
| | #**Cover with foil and leave undisturbed for 1-2 hours<br> |
| | #Wash 3X 15min in PBS. |
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| *[http://blast.ncbi.nlm.nih.gov/Blast.cgi NCBI Blast]
| | Cell can be imaged in PBS, or mounted using DAKO on a slide. Imaging is generally better in PBS, as DAKO can distort cells as it dries. |
| *[http://ca.expasy.org/ ExPASy Tools]<br>
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| *[http://www.basic.northwestern.edu/biotools/oligocalc.html OligoCalc] - PCR primer Tm calculator<br>
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| *[http://tools.neb.com/NEBcutter2/index.php NEB Cutter]<br>
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| *[http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/isoschizomers.asp NEB Isoschizomers]<br>
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| *[http://searchlauncher.bcm.tmc.edu/seq-util/Options/revcomp.html Reverse-Complement DNA Sequence]<br>
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| *[http://www.insilico.uni-duesseldorf.de/Lig_Input.html Ligation Calculator]
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| *[http://www.addgene.org/ AddGene] - clone by e-mail!
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| *[http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_pattinprot.html PattenProt] - search genomes for protein patterns
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| *[http://workbench.sdsc.edu/ Biology Workbench]<br>
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| *[http://www.ebi.ac.uk/Tools/sequence.html EMBL Sequence Tools]<br>
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| *[http://www.ncbi.nlm.nih.gov/projects/gorf/ NCBI ORF Finder]<br>
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| *[http://searchlauncher.bcm.tmc.edu/ BCM Search Launcher]<br>
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| *[http://swissmodel.expasy.org/ SWISS Model protein modeling]<br>
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| *[http://searchlauncher.bcm.tmc.edu/seq-search/struc-predict.html Secondary Structure Prediction]<br>
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| *[http://www.predictprotein.org/ PredictProtein] - Protein structure prediction<br>
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| *[http://3d-alignment.eu/ STRAP] - Protein aligments with structure<br>
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| <br> | | <br> |
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| ===== ''In Vivo'' Tools<br> ===== | | == Recipes == |
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| *[http://www.emouseatlas.org/emap/home.html EMAP] - Virtual mouse anatomy<br>
| | ===== PEM Buffer: ===== |
| *[http://phenome.jax.org/ Mouse phenome database] - mouse phenotypes<br>
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| | * 80 mM PIPES pH 6.8 |
| ===== Microscopy Tools =====
| | * 5 mM EGTA |
| | * 2 mM MgCl<sub>2</sub> |
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| *[http://www.microscopyu.com/ Microscopy U] - Everything you want to know about microscopes<br>
| | ===== Antibody Buffer: ===== |
| *[http://jcb.rupress.org/content/166/1/11.full Paper on Image Processing Standards] - How not to loose your job
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| *[http://www.invitrogen.com/site/us/en/home/support/Research-Tools/Fluorescence-SpectraViewer.html Fluorophore Spectra Viewer] at Life Technology<br>
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| *[http://www.mcb.arizona.edu/ipc/fret/ Fluorescent Spectra Database] - FRET and other
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| *[http://www.mcb.arizona.edu/IPC/spectra_page.htm Yet More Spectra] - from Arizona University
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| *[http://www.confocal-microscopy.org/ www.confocal-microscopy.org] - Data on LSM methods and equipment<br>
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| *[http://fiji.sc/wiki/index.php/Fiji FIJI] - <u>FREE</u> imageJ based image processing program
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| *[http://www.dspguide.com/pdfbook.htm Free] image processing textbook
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| *[http://www.archive.org/details/Lectures_on_Image_Processing Lectures on image processing]<br>
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| *[http://vaa3d.org/ VAA3D] FREE viewer for large 3/4/5D datasets
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| <br>
| | PBS + 0.1% triton X-100 + 5% donkey serum or 2.5% BSA (use goat serum of secondary is derived in goats). |
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| ===== Protease Tools =====
| | ''For 10ml:'' |
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| *[http://merops.sanger.ac.uk/ MEROPS] - Peptidase database | | *9.5ml PBS |
| *[http://www.proteolysis.org/proteases PMAP] - Proteolysis Map | | *10ul Triton X-100 (exclude if surface-staining) |
| *[http://casbase.org/casvm/index.html CASVM] - Caspace substrate prediction
| | *500ul serum or 250mg BSA |
| *[http://bioinf.gen.tcd.ie/casbah/ CASBAH] - Caspase cleaveage site database | |
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| ===== Lipid Tools =====
| | Note: 1% BSA can be used in place of serum in many cases, especially after step 3. |
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| *[http://www.lipidmaps.org/ Lipid Maps] - Lipidomics gateway at ''Nature''
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| *[http://www.avantilipids.com/ Avanti Lipids] - Buy lipids
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| <br> | | <br> |
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| ===== Chemical Tools ===== | | == Working Conditions<br> == |
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| *[http://www.chemspider.com/ ChemSpider] - General chemistry database
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| *[http://pubchem.ncbi.nlm.nih.gov/ PubChem] - Chemical structure database
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| <br>
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| <br> | |
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| | {| width="100%" cellspacing="1" cellpadding="1" border="1" |
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| | | '''Antibody<br>''' |
| | | '''Fixation'''<br> |
| | | '''Primary Conditions<br>''' |
| | | '''Washes'''<br> |
| | | '''Secondary Conditions'''<br> |
| | | '''Washes<br>''' |
| | |- |
| | | 4G10<br> |
| | | 4% PFA, 20', 4C<br> |
| | | 1:100 in antibody buffer, overnight, 4C<br> |
| | | 3 x 15min PBS<br> |
| | | 1:200 anti-mouse in antibody buffer, 1 to 2 hours, room temp.<br> |
| | | 3 x 15min PBS<br> |
| | |- |
| | | 4G10<br> |
| | | 4% PFA, 20', 4C<br> |
| | | 1:100 in antibody buffer, 4hrs, 20C<br> |
| | | 3 x 15min PBS<br> |
| | | 1:200 anti-mouse in antibody buffer, 1 to 2 hours, room temp.<br> |
| | | 3 x 15min PBS<br> |
| | |- |
| | | THE His6 Antibody ([http://www.genscript.com/antibody/A00186-THE_sup_TM_sup_His_Tag_Antibody_mAb_Mouse.html GenScript A00186])<br> |
| | | 4% PFA, 20', 4C<br> |
| | | 1:250-1:500 in antibody buffer, 1-4hrs, 20C<br> |
| | | 3 x 15min PBS<br> |
| | | 1:1000 anti-mouse in antibody buffer, 1 to 2 hours, room temp.<br> |
| | | 3 x 15min PBS<br> |
| |} | | |} |
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