Western Blotting

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This is a generic protocol for western blotting.  Optimized procedures for specific blots are listed at the end of this protocol.

Contents

Cell Preparation and Lysis

  1. Prepare cells for lysis.  This may involve activating the cells with an antibody or chemical, treatment with an inhibitor, serum starvation, reducing focal contact signaling, etc.
  2. Wash cells with PBS or other media to remove serum, cell debris and other contaminants.
Gentle Lysis:
  1. Add a minimal volume of ice-cold lysis buffer (300ul for 35mm plate, 1ml for 150mm plate), and use a cell scraper to suspend cells in lysis buffer.
  2. Aspirate with pipettor, repetitively pipette to breakup clumps
  3. Transfer to a 1.5ml tube, and place on ice.  Rotate at 4C for 40min to 1hr to complete lysis
  4. Minifuge at 4C, 13,000 RPM for 15min to pellet DNA and insoluble material.  Transfer supernatant to clean 1.5ml tube.
  5. Add 1/4th volume of 4X Lamelis or 1/6th volume 4x or 6x Laemmli's buffer with β-mercaptoethanol.
Rapid Lysis:
  1. Dilute laemmli's + β-mercaptoethanol to 1X concentration in lysis buffer.
  2. Add protease and/or phosphatase inhibitors, as needed.
  3. Add a minimal volume of the buffer prepared in step 1 to the plate (300ul for 35mm plate, 1ml for 150mm plate), and use a cell scraper to suspend cells.
  4. Vortex or sonicate sample to shear DNA

After either type of lysis, samples should be immediately boiled for 5 minutes.

Casting of Acrylamide Gels

  1. Clean glass with 70% ethanol and dry with lint-free clot (kimwipe).
  2. Place glass plates into green clamp, and mount on casting stand.  Mark glass at the top of the square section of the clamp, ~5cm from top of gel.
  3. Mix lower (resolving) gel as per chart, without adding TEMED. Use higher % gel for separating smaller proteins, lower % gel for larger. Most studies can use the 10% gel.
  4. Place 10ul TEMED in the bottom of a 1.5ml tube.  Quickly add 1ml of the separating (lower) gel mix put 500ul (1.5mm gels) or 250ul (0.75mm gels) into each gel.  Do this by pouring the gel down one side of the glass plates, and tip gel to the side to spread evenly along the bottom (~5min). This forms a plug. Gel will harden in less then 1 minute, so move quickly.
  5. To remaining lower buffer mix add TEMED as per chart, swirl to mix, and pour gel (pipette down the side to prevent bubbles). Pour until gel is ~2mm above mark made on glass (~10ml for 1.5mm gels). Cover with a small volume of H2O-saturated butenol or isopropyl alcohol. Let harden (~30min). When hard there will be a double-interface between the gel - an excreted water layer plus the butenol layer.
  6. Pour off butenol layer, and rinse 2-3 times with ddH2O. Dry with blotting paper.
  7. Place comb into apparatus. Make up upper (stacking) mix, and pour immediately. Pour until a thin layer of gel is on edge of glass (~35 min). Bang gel to remove any bubbles.
  8. Once hardened remove comb. Place gel in running apparatus, put in running buffer, and then use a syringe to clear out the wells with running buffer.
Lower (resolving) gel, 2 gels (20ml)
8%
10%
12%
14%
16%
18%
20%

30% acrilimide (mls)

5.3ml
6.6ml
8.0 ml
9.4 ml
10.7 ml
12.0 ml
13.3 ml

dH2O

9.3ml
8ml
6.6ml
5.4ml
4.1ml
2.8ml
0.5ml

4X Resolving Lower Gel Buffer (10X tris, pH 8.8)


5ml

20% SDS


100ul

10% APS


100ul

TEMED


10ul


Upper (stacking) gel
2 gels
4 gels

30% acrilimide (mls)

1.3ml
2.6ml

dH2O

6.1ml
12.2ml

4X Stacking Upper Gel Buffer (10X tris, pH 6.8)

2.5ml
5ml

20% SDS

50ul
100ul

10% APS

50ul
100ul

TEMED

10ul
20ul


Loading, Running and Transferring the Gel

Loading & Running the Gel
  1. Using long tips, carefully load the desired amount of cell lysate to each well.  Generally 5ul-30ul will be required per lane, per blot.  Load ladder as needed; generally 2ul is sufficient for developing on the licore, 5-8ul is generally required for conventional blots.
  2. Top off interior reservoir with running buffer
  3. Run gel at 80-150V; lower voltages will generally produce cleaner bands, while higher voltages will run faster.  Voltages above 125V should be avoided, due to the risk of melting the gel.
  4. Run gel until blue dye nears the bottom of the gel (time varies with voltage)
Note: if looking for small proteins stop gel when blue band is 2/3rds the way down the gel, as to avoid running small proteins off of gel.
Transferring the Gel

Note: Wear gloves and handle membrane with tweezers; proteins can transfer from you skin and ruin the blot!

  1. Cut 2 pieces of blotting paper and nitrocellulose or PDMS membrane ~10% larger than the resolving section of the gel.
  2. Wet nitrocellulose with transfer buffer.  PDMS must be whetted 2min in pure methanol, followed by 2min in transfer buffer, before use.
  3. Extract gel from glass plates.  Cut off stacking gel, plug, and any unused wells.
  4. Wash gel in transfer buffer.
  5. Assemble transfer cassette:
  6. White plate
    • Blotting paper
    • Membrane
    • Gel
    • Blotting paper
    • Black plate
  7. Insert "sandwich" into transfer apparatus, with the black plate facing the black wall of the transfer apparatus. Put stirbar and ice pack into chamber and fill with transfer buffer.
  8. Bang transfer apparatus to remove any air bubbles.
Note: foam spacers can be placed between the plates and the blotting paper to secure "sandwich" in transfer cassette.

Warning: do not force cassette shut - you will squish/destroy the gel.  If the fit is tight, remove a foam spacer or use thinner blotting paper.
Quick Transfer
  1. Put chamber into an ice bucket with a layer of ice on the bottom, and pack ice around chamber
  2. Run at 85V for 2 hours, stirring on stir plate
Overnight Transfer
  1. Transfer chamber/stirplate to walk-in fridge
  2. Run gel at 40-50V overnight, stirring on stir-plate

Immunoblotting

  1. Block membrane on a shaker for at least 2hrs at 20C with TBST plus 5% albumin or 5% milk powder.  Albumin should be used for phospho-blots, as milk powder contains phosphatases of phospho-proteins.  Block can also be over night at 4C.
  2. Seal membrane in a vaccu-seal bag, leaving one side open.  Seal bag as close to membrane as possible
  3. Add 3-5ml of primary antibody at desired concentration (usually 1:100-1:1000), in the same buffer gel was blocked in.  Seal remaining side of bag.
  4. Incubate at the desired temperature for the desired length of time.  Common values are:
    • 2 to 4 hours at 20C
    • Overnight at 4C
  5. Wash 3-5 times, 15min/wash, with TBST.  More/longer washes reduce background, but can reduce signal.
  6. In wash tray add 10ml of 1:10,000 (HRP-conjugated) or 1:15,000 (licore-conjugated) secondary antibody, diluted in blocking buffer.  Incubate on shaker, 20C for 1 hour.
  7. Wash 3-5 times, 15min/wash, with TBST. More/longer washes reduce background, but can reduce signal.
  8. Image membrane using substrate and X-ray file (HRP secondaries only) or licore.

Recipes

Lysis Buffer
Reagent
Stock Solution
Volume
ddH2O
--
7.68ml
Tris, pH 8.0
20mM
200ul
NaCl
0.15M
750ul
EDTA
2mM
40ul
NP40
--
100ul
Glycerol
--
1000ul
Na3VO4
1mM
100ul

The above recipe is for 10ml.  This buffer can be made in bulk and used as a wash buffer, or allotted and frozen for future use.  Immediately before use add protease inhibitors at the manufacturers recommended concentration, and phosphatase inhibitors if required.

Laemmli's Buffer, 4x
  • 2.4 ml 1 M Tris pH 6.8 (Same as upper gel buffer)
  • 0.8 g SDS stock
  • 4 ml 100% glycerol
  • 0.01% bromophenol blue. Final Concentration is .02%
  • 2.8 ml ddH2O

Before use add 1/10th volume of β-mercaptoethanol

Laemmli's Buffer, 6x
  • 1.2g SDS (sodium dodecyl sulfate)
  • 0.01% bromophenol blue
  • 4.7ml glycerol
  • 1.2ml Tris 0.5M pH6.8
  • 2.1ml ddH2O

Before use add 1/8th volume of β-mercaptoethanol

4x Lower Gel Buffer
  • 182g Tris-Base (1.5M)
  • 1L dH2O

pH to 8.8; do not overshoot. Use glass pipettes to pH.

4x Upper Gel Buffer
  • 30.28g Tris-HCl (0.5M)
  • 500ml dH2O

pH to 6.8, do not overshoot. Use glass pipettes.

10x Running Buffer
  • 30.4g Tri-HCl
  • 144.2g Glycine
  • 10g SDS

Dissolve in 1L dH2O. Dilute 1:10 in ddH2O for use as running buffer

10X Transfer Buffer (Towbin Buffer)
  • 30.3g Tris-Base
  • 144.15g Glycine
  • 100ml 10% SDS (0.1%)

Bring upto 1L in ddH2O.

1X Transfer Buffer (Towbin Buffer)
  • 100ml 10X buffer
  • 200ml methanol
  • 700ml ddH2O
10x TBS
  • 302.5g Tris-Base
  • 400g NaCl
  • 18g KCl

Dilute to 5L in dH2O, pH to 7.5 with saturated HCl.

Wash Buffer (TBST)

to 1X TBS ADD:

Standard
0.1% Tween-20
Strong
0.1% Tween-20 + 0.1% NP-40
Extra-Strong
0.1% Tween-20 + 1.5% NP-40
Note: Standard buffer is for most application. Strong/extra strong are only for antibodies with a high degree of non-specificity.

Optimized Protocols

Antibody
Primary
Wash
Secondary
Wash
4G10 (anti-phosphotyrosine)
1:1000 to 1:1500 in TBST + 5% BSA.  2 hours at room temp.
3x15min TBST
1:15,000 anti-mouse licore in TBST + 5% BSA.  1 hour at room temp
3x15min TBST
Actin (clone 1A4, ascites)
1:500 in TBST + 5% dry milk powder.  2 hours at room temp.
3x15min TBST
1:15,000 anti-mouse licore in TBST + 5% BSA. 1 hour at room temp
3x15min TBST
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