Western blot protocols

A western blot experiment, or western blotting, is a routine technique for protein analysis. The specificity of the antibody-antigen interaction enables a target protein to be identified in the midst of a complex protein mixture. Here, you can find a collection of western blot recipes for commonly used protein electrophoresis and western blot buffers and stock solutions, and general western blotting protocols for chemiluminescent and fluorescent detection to guide you through your experiment.

If you find this doesn’t work for your specific protein of interest, try our BlotBuilder Product Selection Tool to get a set of recommended products with a personalized western blot protocol.

Western blot protocol for chemiluminescent detection

View recommended buffer formulations under Buffer Recipes tab. Download a personalized editable version of this chemiluminescent protocol.

Materials

  • Nitrocellulose or PVDF transfer membrane (e.g. Thermo Scientific membranes, Cat. No. 88018 or 88518, or equivalent)
  • Transfer buffer (e.g. NuPAGE Transfer Buffer, Cat. No. NP0006, Novex Tris-Glycine Transfer Buffer, Cat. No. LC3675)
  • Wash buffer (Tris-buffered or phosphate-buffered saline with 0.05% Tween 20, Cat. No. 28360 or 28352)
  • Blocking buffer (e.g. Pierce Clear Milk Blocking Buffer, Cat. No. 37587)
  • Incubation trays and containers
  • Primary antibodies (e.g. Invitrogen western blot validated primary antibodies)
  • Secondary antibodies (e.g. Invitrogen western blot validated HRP antibodies)
  • Chemiluminescent HRP substrate (e.g. SuperSignal West Pico PLUS or SuperSignal West Atto Ultimate Sensitivity Substrate, Cat. No. 34580 or A38555)

Protocol

  1. Prepare transfer buffer for wet and semi-dry transfers based on gel chemistry.
  1. Prepare transfer membrane (semi-dry or wet transfers). Follow manufacture instructions for dry membrane preparations.
    • PVDF: pre-wet in methanol or ethanol (100%) for 30 seconds, briefly rinse in deionized water, and equilibrate in transfer buffer for 5 minutes.
    • Nitrocellulose: equilibrate directly in transfer buffer for 5 minutes.
  1. Follow manufacture instructions for wet, semi-dry, or dry transfer.
  1. After protein transfer, wash the membrane in deionized water 4 times for 5 minutes each with agitation to remove all transfer buffer.
  1. Incubate the membrane with a sufficient volume of blocking buffer for 30–60 minutes at room temperature with agitation.
  1. Dilute the primary antibody per supplier recommendations in the blocking buffer.

Recommended primary antibody dilutions to use with Thermo Scientific chemiluminescent substrates.

  Pierce ECL SuperSignal West Pico Plus SuperSignal West Dura SuperSignal West Femto SuperSignal West Atto
Recommended primary antibody dilutions 1:1,000 (0.2–10 µg/mL) 1:1,000 (0.2–1.0 µg/mL) 1:5,000 (0.02–1.0 µg/mL) 1:5,000 (0.01–0.2 µg/mL) 1:5,000 (0.2–1.0 µg/mL)
  1. Incubate the membrane protein-side up in the primary antibody solution with agitation, for 1 hour at room temperature or overnight at 2–8°C. Ensure the volume of the antibody solution is enough to fully cover the membrane.
  1. Wash the membrane 3 times with agitation for 10 minutes each in wash buffer.
  1. Prepare dilutions of the conjugated secondary antibody in appropriate volume of wash buffer or alternatively in blocking buffer.

Protocol tips

At Step 8, if using an enzyme-conjugated primary antibody, proceed to Step 13.

Recommended secondary antibody dilutions to use with Thermo Scientific chemiluminescent substrates.

  Pierce ECL SuperSignal West Pico Plus SuperSignal West Dura SuperSignal West Femto SuperSignal West Atto
Recommended secondary antibody dilutions 1:1,000 - 1:15,000 (0.07–1.0 µg/mL) 1:20,000 - 1:100,000 (10–50 ng/mL) 1:50,000 - 1:250,000 (4–20 ng/mL) 1:100,000 - 1:500,000 (2–10 ng/mL) 1:100,000 - 1:250,000 (4–10 ng/mL)
  1. Incubate the membrane protein-side up in the secondary antibody solution for 1 hour with agitation at room temperature. Ensure the volume of the antibody solution is enough to fully cover the membrane.
  1. Wash the membrane 6 times with agitation for 5 minutes each in wash buffer to remove any unbound secondary antibodies. It is crucial to thoroughly wash the membrane at this step.
  1. Prepare working solution of chemiluminescent substrate based upon manufacture instruction. Suggested volume of ~8–10 mL for mini blots and 15 mL for midi blots (0.1 mL working solution per cm2 of membrane).
  1. Incubate the blot with the working solution for 1 min. when using standard ECL substrates or 5 min. when using high-performance substrates, such as SuperSignal substrates.
  1. Remove the blot from working solution and drain excess reagent.
  1. Place the blot in clear plastic wrap or sheet protector and remove bubbles by rolling with blot roller or glass pipette.
  1. Image the blot using film or appropriate imaging system.

Fluorescent western blotting protocol

View recommended buffer formulations under Buffer Recipes tab. Download a personalized editable version of this fluorescent protocol.

Materials

  • Nitrocellulose or PVDF transfer membrane (e.g. Thermo Scientific membranes, Cat. No. 88018 or 22860, or equivalent)
  • Transfer buffer (e.g. NuPAGE Transfer Buffer, Cat. No. NP0006, Novex Tris-Glycine Transfer Buffer, Cat. No. LC3675)
  • Wash buffer (Tris-buffered or phosphate-buffered saline with 0.05% Tween 20, Cat. No. 28360 or 28352)
  • Filtered blocking buffer (e.g. Blocker FL Fluorescent Blocking Buffer, Cat. No. 37565)
  • Incubation trays and containers
  • Primary antibodies (e.g. Invitrogen western blot validated primary antibodies)
  • Secondary antibodies (e.g. Invitrogen fluorescently labeled highly cross-absorbed secondary antibodies)

Protocol

  1. Prepare transfer buffer for wet and semi-dry transfers based on gel chemistry.
  1. Prepare transfer membrane (semi-dry or wet transfers). Follow manufacture instructions for dry membrane preparations.
    • PVDF: pre-wet in methanol or ethanol (100%) for 30 seconds, briefly rinse in deionized water, and equilibrate in transfer buffer for 5 minutes.
    • Nitrocellulose: equilibrate directly in transfer buffer for 5 minutes.
  1. Follow manufacture instructions for wet, semi-dry, or dry transfer.
  1. After protein transfer, wash the membrane in deionized water 4 times for 5 minutes each with agitation to remove all transfer buffer.
  1. Incubate the membrane with a sufficient volume of blocking buffer for 30–60 minutes at room temperature with agitation.
  1. Dilute the primary antibody per supplier recommendations in the blocking buffer.
  1. Incubate the membrane protein-side up in the primary antibody solution with agitation, for 1 hour at room temperature or overnight at 2–8°C. Ensure the volume of the antibody solution is enough to fully cover the membrane.
  1. Wash the membrane 3 times with agitation for 10 minutes each in wash buffer. If using a fluorescently conjugated primary antibody, proceed to Step 11.
  1. Prepare dilutions of the conjugated secondary antibody to 0.4 to 0.1 µg/mL in appropriate volume of wash buffer or alternatively in blocking buffer. From a 2 mg/mL antibody stock, dilute 1:5,000 to 1:20,000:
    • 1:5,000: 3 µL of secondary antibody in 15 mL wash buffer
    • 1:10,000: 1.5 µL of secondary antibody in 15 mL wash buffer
    • 1:20,000: 0.75 µL of secondary antibody in 15 mL wash buffer
  1. Incubate the membrane protein-side up in the secondary antibody solution for 1 hour with agitation at room temperature. Ensure the volume of the antibody solution is enough to fully cover the membrane and protect the membrane from bright light to prevent photobleaching of the fluorescent dyes.
  1. Wash the membrane 6 times with agitation for 5 minutes each in wash buffer to remove any unbound secondary antibodies. It is crucial to thoroughly wash the membrane at this step.
  1. Blots can be imaged immediately while still wet, or alternatively may be dried prior to imaging. Place each blot in a sheet protector or on a clean surface prior to imaging to prevent contamination.
  1. Image the blot using an appropriate imaging system with fluorescence detection mode.

Protocol tips

Do not add detergent to blocking buffer, as this may increase background fluorescence.

For typical incubation trays, use at least 15 mL for mini blots and 30 mL for midi blots to fully cover the membrane. Avoid low volumes, as differences in agitation and coverage can produce high or uneven background.

Protocol tips

The final wash time may be reduced by filling and decanting the tray with distilled water 4 times, then moving forward with three 5-minute washes in wash buffer.

Protocol tips

To dry the membrane, place it between two sheets of western blot filter paper to protect it from light exposure while drying. Drying the membrane allows for extended storage of the blot and can reduce exposure times. Store blots in the dark to prevent photobleaching.

Western Blot Buffer Recipes

Stock solutions

  • 1 M Tris-HCl, pH 7.6 (100 mL)
  • 0.5 M Tris-HCl, pH 6.8 (100 mL)
  • 10% SDS (10 mL)
  • 1.0% Bromophenol Blue (10 mL)
  • 10X Tris Buffered Saline (TBS)
  • 10X Phosphate Buffered Saline (PBS)

Sample preparation buffers

  • RIPA buffer
  • 2X Tris-Glycine SDS Sample buffer (Laemmli buffer)
  • 4X LDS Sample Buffer

Electrophoresis running buffers

  • 10X Tris-Glycine SDS Running Buffer
  • 10X Tris-Glycine Native Running Buffer
  • 20X MOPS SDS Running Buffer
  • 20X MES SDS Running Buffer
  • 10X Tricine SDS Running Buffer

Transfer buffer

  • 25X Tris-Glycine Transfer Buffer
  • 20X Bis-Tris Transfer Buffer

Wash buffers

  • Tris-buffered saline with Tween 20 (TBST)
  • Phosphate buffered saline with Tween 20 (PBST)

Blocking and stripping buffers recipes

  • 5% Nonfat Milk
  • 3% BSA
  • Stripping Buffer

Gel casting recipes

  • SureCast Reagents
  • Standalone Reagents

Stock solutions

1 M Tris-HCl, pH 7.6 (100 mL)

Tris Base 12.11 g
Deionized water 80 mL
Adjust pH to 7.6 with HCl
Deionized water to 100 mL

0.5 M Tris-HCl, pH 6.8 (100 mL)

Tris Base 6.06 g
Deionized water 60 mL
Adjust pH to 6.8 with HCl
Deionized water to 100 mL

10% SDS (10 mL)

SDS 1.00 g
Deionized water to 10 mL

1.0% Bromophenol Blue (10 mL)

Bromophenol blue 100 mg
Deionized water to 10 mL

10X Tris Buffered Saline (TBS)

Tris Base 24 g
NaCl 88 g
Deionized water 900 mL
pH to 7.6 with HCl
Deionized water to 1000 mL

10X Phosphate Buffered Saline (PBS)

NaCl 80 g
KCl 2 g
Na2HPO4 14.4 g
NaH2PO4 2.4 g
Deionized water 900 mL
pH to 7.0 with NaOH
Deionized water to 1000 mL

Sample preparation buffers

RIPA buffer: 25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS (100 mL)

NaCl 0.88 g
NP-40 1 g
Sodium deoxycholate 1 g
10% SDS 1 mL
1 M Tris-HCl, pH 7.6 2.5 mL
Deionized water to 100 mL
Protease Inhibitor Tablet (Cat. No. A32965) 2 tablets

SDS Sample buffer (Laemmli buffer): 63 mM Tris HCl, 10% Glycerol, 2% SDS, 0.0025% Bromophenol Blue, pH 6.8 (10 mL)

Recipe for 2X buffer stock:

0.5 M Tris-HCl pH 6.8 2.5 mL
Glycerol 2 mL
10% (w/v) SDS 4 mL
0.1% (w/v) Bromophenol Blue 0.5 mL
Deionized water to 10 mL

The buffer is stable for 6 months when stored at 4°C.

LDS Sample Buffer: 106 mM Tris HCl, 141 mM Tris Base, 2% LDS, 10% Glycerol, 0.51 mM EDTA, 0.22 mM SERVA Blue G250, 0.175 mM Phenol Red, pH 8.5.

Recipe for 4X buffer stock:

Tris HCl 0.666 g
Tris Base 0.682 g
LDS 0.800 g
EDTA 0.006 g
Glycerol 4 g
SERVA Blue G250 (1% solution) 0.75 mL
Phenol Red (1% solution) 0.25 mL
Deionized water to 10 mL

The buffer is stable for 6 months when stored at 4°C. Do not use acid or base to adjust pH.

Electrophoresis running buffers

Tris-Glycine SDS Running Buffer: 25 mM Tris Base, 192 mM Glycine, 0.1% SDS, pH 8.3.

Recipe for 10X buffer stock:

Tris Base 29 g
Glycine 144 g
SDS 10 g
Deionized water to 1000 mL

Tris-Glycine Native Running Buffer: 25 mM Tris Base, 192 mM Glycine, pH 8.3.

Recipe for 10X buffer stock:

Tris Base 29 g
Glycine 144 g
Deionized water to 1000 mL

MOPS SDS Running Buffer: 50 mM MOPS, 50 mM Tris Base, 0.1% SDS, 1 mM EDTA, pH 7.7.

Recipe for 20X buffer stock:

MOPS 104.6 g
Tris Base 60.6 g
SDS 10 g
EDTA 3.0 g
Deionized water to 500 mL

Do not use acid or base to adjust pH.

MES SDS Running Buffer: 50 mM MES, 50 mM Tris Base, 0.1% SDS, 1 mM EDTA, pH 7.3.

Recipe for 20X buffer stock:

MES 97.6 g
Tris Base 60.6 g
SDS 10 g
EDTA 3.0 g
Deionized water to 500 mL

Do not use acid or base to adjust pH.

Tricine SDS Running Buffer: 100 mM Tris Base, 100 mM Tricine, 0.1% SDS, pH 8.3.

Recipe for 10X buffer stock:

Tris Base 121 g
Tricine 179 g
SDS 10 g
Deionized water to 1000 mL

The buffer is stable for 6 months when stored at room temperature.
Do not use acid or base to adjust pH.

Transfer buffer recipes

Tris-Glycine Transfer Buffer: 12 mM Tris Base, 96 mM Glycine, pH 8.3.

Recipe for 25X buffer stock:

Tris Base 18.2 g
Glycine 90 g
Deionized water to 500 mL

Bis-Tris Transfer Buffer: 25 mM Bicine, 25 mM Bis-Tris (free base), 1 mM EDTA, pH 7.2.

Recipe for 20X buffer stock:

Bicine 10.2 g
Bis-Tris (free base) 13.1 g
EDTA 0.75 g
Deionized water 125 mL

The buffer is stable for 6 months when stored at 4°C.
Do not use acid or base to adjust pH.

Wash buffers recipes

Tris-buffered saline with Tween 20 (TBST)

10X TBS 100 mL
Tween 20 1 mL
Deionized water to 1000 mL

Phosphate buffered saline with Tween 20 (PBST)

10X TBS 100 mL
Tween 20 1 mL
Deionized water to 1000 mL

Blocking and stripping buffers recipes

5% nonfat milk

Nonfat dry milk 2.5 g
TBST or PBST Up to 50 mL
Filter to remove particulates  

3% BSA

BSA 1.5 g
TBST or PBST Up to 50 mL
Filter to remove particulates  

Stripping buffer

0.5 M Tris HCl, pH 6.8 12.5 mL
10% SDS 20 mL
2-mercaptoethanol 0.8 mL
Deionized water 67.5 mL

Gel casting recipes

Recipes with SureCast reagents

The volumes provided in the table are for a single gel. Scale volumes proportionally based on the number of gels to be cast.

  Polyacrylamide %
Solution 4% 6% 8% 10% 12% 14% 16% 18% 20%
SureCast Acrylamide (40%) 0.8 mL 1.2 mL 1.6 mL 2.0 mL 2.4 mL 2.8 mL 3.3 mL 3.6 mL 4.0 mL
SureCast Resolving Buffer 2.0 mL 2.0 mL 2.0 mL 2.0 mL 2.0 mL 2.0 mL 2.0 mL 2.0 mL 2.0 mL
Distilled water 5.1 mL 4.7 mL 4.3 mL 3.9 mL 3.5 mL 3.1 mL 2.7 mL 2.3 mL 1.9 mL
10% SureCast APS 80 µL 80 µL 80 µL 80 µL 80 µL 80 µL 80 µL 80 µL 80 µL
SureCast TEMED* 8 µL 8 µL 8 µL 8 µL 8 µL 8 µL 8 µL 8 µL 8 µL

*Add this last and mix well just before the gel is to be poured

Prepare stacking gel solution according to the following table. The volumes provided in the table are for a single gel. Scale volumes proportionally based on the number of gels to be cast. Note: Solutions do not require degassing.

Solution 4%
SureCast Acrylamide (40%) 0.30 mL
SureCast Stacking Buffer 0.75 mL
Distilled water 1.92 mL
10% SureCast APS 30 µL
SureCast TEMED* 3 µL

*Add this last and mix well just before the gel is to be poured

Recipes with standalone reagents

Stock solutions

Prepare the following stock solutions: all solutions can be stored at room temperature.

50% Acrylamide/BIS (29:1)
  • 48.3 g Acrylamide
  • 1.7 g BIS
Bring to 100 mL with water.
Store up to two months in a dark glass bottle.
Separating Gel Buffer (1 M Tris-HCl, pH 8.8)
  • Add 30.3 g Tris to 150 mL water
  • Adjust pH 8.8 with HCL
Bring to 250 mL with water.
Stacking Gel Buffer (0.375M Tris HCl, pH 6.8)
  • Add 11.4 g Tris to about 150 mL water
  • Adjust to pH 6.8 with HCl
Bring to 250 mL with water.
Catalyst-Ammonium Persulfate (Make fresh the day of use)
  • 100 mg Ammonium Persulfate
Bring to 2 mL in water.
10% SDS
  • 10.0 g SDS
Bring to 100 mL with water.
50% Sucrose
  • 50.0 g Sucrose
Bring to 100 mL with water.
   

Separating gel

The following recipes are for approximately 25 mL of separating gel, enough for four 1.0-mm thick mini gels. Scale volumes proportionally based on the number of gels to be cast.

Solution 6% Gel 8% Gel 10% Gel 12% Gel 14% Gel 16% Gel 18% Gel 20% Gel
50% Acrylamide/BIS 3.0 mL 4.0 mL 5.0 mL 6.0 mL 7.0 mL 8.0 mL 9.0 mL 10.0 mL
Separating Gel Buffer 9.4 mL 9.4 mL 9.4 mL 9.4 mL 9.4 mL 9.4 mL 9.4 mL 9.4 mL
10% SDS 250 µL 250 µL 250 µL 250 µL 250 µL 250 µL 250 µL 250 µL
50% Sucrose* 4.0 mL 4.0 mL 4.0 mL 4.0 mL 4.0 mL 4.0 mL 4.0 mL 4.0 mL
Water 7.8 mL 6.8 mL 5.8 mL 4.8 mL 3.7 mL 2.7 mL 1.7 mL 750 µL
TEMED** 6.25 µL 6.25 µL 6.25 µL 6.25 µL 6.25 µL 6.25 µL 6.25 µL 6.25 µL
Catalyst** 625 µL 625 µL 625 µL 625 µL 625 µL 625 µL 625 µL 625 µL

*Optional but recommended because it makes it easy to form a good interface between the separating gel and the overlay. If omitted, increase the amount of water added to make up for the volume of the sucrose solution (increase the water by 4.0 mL for the above tables).
**Add these last and mix well just before the gel is to be poured.

Stacking gel

Following recipe is for 4% Stacking Gel (12.5 mL)

Solution 4%
50% Acrylamide/BIS 1.0 mL
Stacking Gel Buffer 4.2 mL
10% SDS 125 µL
Water 6.3 mL
TEMED* 5.0 µL
Catalyst* 1.0 mL

*Add these last and mix well just before the gel is to be poured.

Western blot protocol for chemiluminescent detection

View recommended buffer formulations under Buffer Recipes tab. Download a personalized editable version of this chemiluminescent protocol.

Materials

  • Nitrocellulose or PVDF transfer membrane (e.g. Thermo Scientific membranes, Cat. No. 88018 or 88518, or equivalent)
  • Transfer buffer (e.g. NuPAGE Transfer Buffer, Cat. No. NP0006, Novex Tris-Glycine Transfer Buffer, Cat. No. LC3675)
  • Wash buffer (Tris-buffered or phosphate-buffered saline with 0.05% Tween 20, Cat. No. 28360 or 28352)
  • Blocking buffer (e.g. Pierce Clear Milk Blocking Buffer, Cat. No. 37587)
  • Incubation trays and containers
  • Primary antibodies (e.g. Invitrogen western blot validated primary antibodies)
  • Secondary antibodies (e.g. Invitrogen western blot validated HRP antibodies)
  • Chemiluminescent HRP substrate (e.g. SuperSignal West Pico PLUS or SuperSignal West Atto Ultimate Sensitivity Substrate, Cat. No. 34580 or A38555)

Protocol

  1. Prepare transfer buffer for wet and semi-dry transfers based on gel chemistry.
  1. Prepare transfer membrane (semi-dry or wet transfers). Follow manufacture instructions for dry membrane preparations.
    • PVDF: pre-wet in methanol or ethanol (100%) for 30 seconds, briefly rinse in deionized water, and equilibrate in transfer buffer for 5 minutes.
    • Nitrocellulose: equilibrate directly in transfer buffer for 5 minutes.
  1. Follow manufacture instructions for wet, semi-dry, or dry transfer.
  1. After protein transfer, wash the membrane in deionized water 4 times for 5 minutes each with agitation to remove all transfer buffer.
  1. Incubate the membrane with a sufficient volume of blocking buffer for 30–60 minutes at room temperature with agitation.
  1. Dilute the primary antibody per supplier recommendations in the blocking buffer.

Recommended primary antibody dilutions to use with Thermo Scientific chemiluminescent substrates.

  Pierce ECL SuperSignal West Pico Plus SuperSignal West Dura SuperSignal West Femto SuperSignal West Atto
Recommended primary antibody dilutions 1:1,000 (0.2–10 µg/mL) 1:1,000 (0.2–1.0 µg/mL) 1:5,000 (0.02–1.0 µg/mL) 1:5,000 (0.01–0.2 µg/mL) 1:5,000 (0.2–1.0 µg/mL)
  1. Incubate the membrane protein-side up in the primary antibody solution with agitation, for 1 hour at room temperature or overnight at 2–8°C. Ensure the volume of the antibody solution is enough to fully cover the membrane.
  1. Wash the membrane 3 times with agitation for 10 minutes each in wash buffer.
  1. Prepare dilutions of the conjugated secondary antibody in appropriate volume of wash buffer or alternatively in blocking buffer.

Protocol tips

At Step 8, if using an enzyme-conjugated primary antibody, proceed to Step 13.

Recommended secondary antibody dilutions to use with Thermo Scientific chemiluminescent substrates.

  Pierce ECL SuperSignal West Pico Plus SuperSignal West Dura SuperSignal West Femto SuperSignal West Atto
Recommended secondary antibody dilutions 1:1,000 - 1:15,000 (0.07–1.0 µg/mL) 1:20,000 - 1:100,000 (10–50 ng/mL) 1:50,000 - 1:250,000 (4–20 ng/mL) 1:100,000 - 1:500,000 (2–10 ng/mL) 1:100,000 - 1:250,000 (4–10 ng/mL)
  1. Incubate the membrane protein-side up in the secondary antibody solution for 1 hour with agitation at room temperature. Ensure the volume of the antibody solution is enough to fully cover the membrane.
  1. Wash the membrane 6 times with agitation for 5 minutes each in wash buffer to remove any unbound secondary antibodies. It is crucial to thoroughly wash the membrane at this step.
  1. Prepare working solution of chemiluminescent substrate based upon manufacture instruction. Suggested volume of ~8–10 mL for mini blots and 15 mL for midi blots (0.1 mL working solution per cm2 of membrane).
  1. Incubate the blot with the working solution for 1 min. when using standard ECL substrates or 5 min. when using high-performance substrates, such as SuperSignal substrates.
  1. Remove the blot from working solution and drain excess reagent.
  1. Place the blot in clear plastic wrap or sheet protector and remove bubbles by rolling with blot roller or glass pipette.
  1. Image the blot using film or appropriate imaging system.

Fluorescent western blotting protocol

View recommended buffer formulations under Buffer Recipes tab. Download a personalized editable version of this fluorescent protocol.

Materials

  • Nitrocellulose or PVDF transfer membrane (e.g. Thermo Scientific membranes, Cat. No. 88018 or 22860, or equivalent)
  • Transfer buffer (e.g. NuPAGE Transfer Buffer, Cat. No. NP0006, Novex Tris-Glycine Transfer Buffer, Cat. No. LC3675)
  • Wash buffer (Tris-buffered or phosphate-buffered saline with 0.05% Tween 20, Cat. No. 28360 or 28352)
  • Filtered blocking buffer (e.g. Blocker FL Fluorescent Blocking Buffer, Cat. No. 37565)
  • Incubation trays and containers
  • Primary antibodies (e.g. Invitrogen western blot validated primary antibodies)
  • Secondary antibodies (e.g. Invitrogen fluorescently labeled highly cross-absorbed secondary antibodies)

Protocol

  1. Prepare transfer buffer for wet and semi-dry transfers based on gel chemistry.
  1. Prepare transfer membrane (semi-dry or wet transfers). Follow manufacture instructions for dry membrane preparations.
    • PVDF: pre-wet in methanol or ethanol (100%) for 30 seconds, briefly rinse in deionized water, and equilibrate in transfer buffer for 5 minutes.
    • Nitrocellulose: equilibrate directly in transfer buffer for 5 minutes.
  1. Follow manufacture instructions for wet, semi-dry, or dry transfer.
  1. After protein transfer, wash the membrane in deionized water 4 times for 5 minutes each with agitation to remove all transfer buffer.
  1. Incubate the membrane with a sufficient volume of blocking buffer for 30–60 minutes at room temperature with agitation.
  1. Dilute the primary antibody per supplier recommendations in the blocking buffer.
  1. Incubate the membrane protein-side up in the primary antibody solution with agitation, for 1 hour at room temperature or overnight at 2–8°C. Ensure the volume of the antibody solution is enough to fully cover the membrane.
  1. Wash the membrane 3 times with agitation for 10 minutes each in wash buffer. If using a fluorescently conjugated primary antibody, proceed to Step 11.
  1. Prepare dilutions of the conjugated secondary antibody to 0.4 to 0.1 µg/mL in appropriate volume of wash buffer or alternatively in blocking buffer. From a 2 mg/mL antibody stock, dilute 1:5,000 to 1:20,000:
    • 1:5,000: 3 µL of secondary antibody in 15 mL wash buffer
    • 1:10,000: 1.5 µL of secondary antibody in 15 mL wash buffer
    • 1:20,000: 0.75 µL of secondary antibody in 15 mL wash buffer
  1. Incubate the membrane protein-side up in the secondary antibody solution for 1 hour with agitation at room temperature. Ensure the volume of the antibody solution is enough to fully cover the membrane and protect the membrane from bright light to prevent photobleaching of the fluorescent dyes.
  1. Wash the membrane 6 times with agitation for 5 minutes each in wash buffer to remove any unbound secondary antibodies. It is crucial to thoroughly wash the membrane at this step.
  1. Blots can be imaged immediately while still wet, or alternatively may be dried prior to imaging. Place each blot in a sheet protector or on a clean surface prior to imaging to prevent contamination.
  1. Image the blot using an appropriate imaging system with fluorescence detection mode.

Protocol tips

Do not add detergent to blocking buffer, as this may increase background fluorescence.

For typical incubation trays, use at least 15 mL for mini blots and 30 mL for midi blots to fully cover the membrane. Avoid low volumes, as differences in agitation and coverage can produce high or uneven background.

Protocol tips

The final wash time may be reduced by filling and decanting the tray with distilled water 4 times, then moving forward with three 5-minute washes in wash buffer.

Protocol tips

To dry the membrane, place it between two sheets of western blot filter paper to protect it from light exposure while drying. Drying the membrane allows for extended storage of the blot and can reduce exposure times. Store blots in the dark to prevent photobleaching.

Western Blot Buffer Recipes

Stock solutions

  • 1 M Tris-HCl, pH 7.6 (100 mL)
  • 0.5 M Tris-HCl, pH 6.8 (100 mL)
  • 10% SDS (10 mL)
  • 1.0% Bromophenol Blue (10 mL)
  • 10X Tris Buffered Saline (TBS)
  • 10X Phosphate Buffered Saline (PBS)

Sample preparation buffers

  • RIPA buffer
  • 2X Tris-Glycine SDS Sample buffer (Laemmli buffer)
  • 4X LDS Sample Buffer

Electrophoresis running buffers

  • 10X Tris-Glycine SDS Running Buffer
  • 10X Tris-Glycine Native Running Buffer
  • 20X MOPS SDS Running Buffer
  • 20X MES SDS Running Buffer
  • 10X Tricine SDS Running Buffer

Transfer buffer

  • 25X Tris-Glycine Transfer Buffer
  • 20X Bis-Tris Transfer Buffer

Wash buffers

  • Tris-buffered saline with Tween 20 (TBST)
  • Phosphate buffered saline with Tween 20 (PBST)

Blocking and stripping buffers recipes

  • 5% Nonfat Milk
  • 3% BSA
  • Stripping Buffer

Gel casting recipes

  • SureCast Reagents
  • Standalone Reagents

Stock solutions

1 M Tris-HCl, pH 7.6 (100 mL)

Tris Base 12.11 g
Deionized water 80 mL
Adjust pH to 7.6 with HCl
Deionized water to 100 mL

0.5 M Tris-HCl, pH 6.8 (100 mL)

Tris Base 6.06 g
Deionized water 60 mL
Adjust pH to 6.8 with HCl
Deionized water to 100 mL

10% SDS (10 mL)

SDS 1.00 g
Deionized water to 10 mL

1.0% Bromophenol Blue (10 mL)

Bromophenol blue 100 mg
Deionized water to 10 mL

10X Tris Buffered Saline (TBS)

Tris Base 24 g
NaCl 88 g
Deionized water 900 mL
pH to 7.6 with HCl
Deionized water to 1000 mL

10X Phosphate Buffered Saline (PBS)

NaCl 80 g
KCl 2 g
Na2HPO4 14.4 g
NaH2PO4 2.4 g
Deionized water 900 mL
pH to 7.0 with NaOH
Deionized water to 1000 mL

Sample preparation buffers

RIPA buffer: 25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS (100 mL)

NaCl 0.88 g
NP-40 1 g
Sodium deoxycholate 1 g
10% SDS 1 mL
1 M Tris-HCl, pH 7.6 2.5 mL
Deionized water to 100 mL
Protease Inhibitor Tablet (Cat. No. A32965) 2 tablets

SDS Sample buffer (Laemmli buffer): 63 mM Tris HCl, 10% Glycerol, 2% SDS, 0.0025% Bromophenol Blue, pH 6.8 (10 mL)

Recipe for 2X buffer stock:

0.5 M Tris-HCl pH 6.8 2.5 mL
Glycerol 2 mL
10% (w/v) SDS 4 mL
0.1% (w/v) Bromophenol Blue 0.5 mL
Deionized water to 10 mL

The buffer is stable for 6 months when stored at 4°C.

LDS Sample Buffer: 106 mM Tris HCl, 141 mM Tris Base, 2% LDS, 10% Glycerol, 0.51 mM EDTA, 0.22 mM SERVA Blue G250, 0.175 mM Phenol Red, pH 8.5.

Recipe for 4X buffer stock:

Tris HCl 0.666 g
Tris Base 0.682 g
LDS 0.800 g
EDTA 0.006 g
Glycerol 4 g
SERVA Blue G250 (1% solution) 0.75 mL
Phenol Red (1% solution) 0.25 mL
Deionized water to 10 mL

The buffer is stable for 6 months when stored at 4°C. Do not use acid or base to adjust pH.

Electrophoresis running buffers

Tris-Glycine SDS Running Buffer: 25 mM Tris Base, 192 mM Glycine, 0.1% SDS, pH 8.3.

Recipe for 10X buffer stock:

Tris Base 29 g
Glycine 144 g
SDS 10 g
Deionized water to 1000 mL

Tris-Glycine Native Running Buffer: 25 mM Tris Base, 192 mM Glycine, pH 8.3.

Recipe for 10X buffer stock:

Tris Base 29 g
Glycine 144 g
Deionized water to 1000 mL

MOPS SDS Running Buffer: 50 mM MOPS, 50 mM Tris Base, 0.1% SDS, 1 mM EDTA, pH 7.7.

Recipe for 20X buffer stock:

MOPS 104.6 g
Tris Base 60.6 g
SDS 10 g
EDTA 3.0 g
Deionized water to 500 mL

Do not use acid or base to adjust pH.

MES SDS Running Buffer: 50 mM MES, 50 mM Tris Base, 0.1% SDS, 1 mM EDTA, pH 7.3.

Recipe for 20X buffer stock:

MES 97.6 g
Tris Base 60.6 g
SDS 10 g
EDTA 3.0 g
Deionized water to 500 mL

Do not use acid or base to adjust pH.

Tricine SDS Running Buffer: 100 mM Tris Base, 100 mM Tricine, 0.1% SDS, pH 8.3.

Recipe for 10X buffer stock:

Tris Base 121 g
Tricine 179 g
SDS 10 g
Deionized water to 1000 mL

The buffer is stable for 6 months when stored at room temperature.
Do not use acid or base to adjust pH.

Transfer buffer recipes

Tris-Glycine Transfer Buffer: 12 mM Tris Base, 96 mM Glycine, pH 8.3.

Recipe for 25X buffer stock:

Tris Base 18.2 g
Glycine 90 g
Deionized water to 500 mL

Bis-Tris Transfer Buffer: 25 mM Bicine, 25 mM Bis-Tris (free base), 1 mM EDTA, pH 7.2.

Recipe for 20X buffer stock:

Bicine 10.2 g
Bis-Tris (free base) 13.1 g
EDTA 0.75 g
Deionized water 125 mL

The buffer is stable for 6 months when stored at 4°C.
Do not use acid or base to adjust pH.

Wash buffers recipes

Tris-buffered saline with Tween 20 (TBST)

10X TBS 100 mL
Tween 20 1 mL
Deionized water to 1000 mL

Phosphate buffered saline with Tween 20 (PBST)

10X TBS 100 mL
Tween 20 1 mL
Deionized water to 1000 mL

Blocking and stripping buffers recipes

5% nonfat milk

Nonfat dry milk 2.5 g
TBST or PBST Up to 50 mL
Filter to remove particulates  

3% BSA

BSA 1.5 g
TBST or PBST Up to 50 mL
Filter to remove particulates  

Stripping buffer

0.5 M Tris HCl, pH 6.8 12.5 mL
10% SDS 20 mL
2-mercaptoethanol 0.8 mL
Deionized water 67.5 mL

Gel casting recipes

Recipes with SureCast reagents

The volumes provided in the table are for a single gel. Scale volumes proportionally based on the number of gels to be cast.

  Polyacrylamide %
Solution 4% 6% 8% 10% 12% 14% 16% 18% 20%
SureCast Acrylamide (40%) 0.8 mL 1.2 mL 1.6 mL 2.0 mL 2.4 mL 2.8 mL 3.3 mL 3.6 mL 4.0 mL
SureCast Resolving Buffer 2.0 mL 2.0 mL 2.0 mL 2.0 mL 2.0 mL 2.0 mL 2.0 mL 2.0 mL 2.0 mL
Distilled water 5.1 mL 4.7 mL 4.3 mL 3.9 mL 3.5 mL 3.1 mL 2.7 mL 2.3 mL 1.9 mL
10% SureCast APS 80 µL 80 µL 80 µL 80 µL 80 µL 80 µL 80 µL 80 µL 80 µL
SureCast TEMED* 8 µL 8 µL 8 µL 8 µL 8 µL 8 µL 8 µL 8 µL 8 µL

*Add this last and mix well just before the gel is to be poured

Prepare stacking gel solution according to the following table. The volumes provided in the table are for a single gel. Scale volumes proportionally based on the number of gels to be cast. Note: Solutions do not require degassing.

Solution 4%
SureCast Acrylamide (40%) 0.30 mL
SureCast Stacking Buffer 0.75 mL
Distilled water 1.92 mL
10% SureCast APS 30 µL
SureCast TEMED* 3 µL

*Add this last and mix well just before the gel is to be poured

Recipes with standalone reagents

Stock solutions

Prepare the following stock solutions: all solutions can be stored at room temperature.

50% Acrylamide/BIS (29:1)
  • 48.3 g Acrylamide
  • 1.7 g BIS
Bring to 100 mL with water.
Store up to two months in a dark glass bottle.
Separating Gel Buffer (1 M Tris-HCl, pH 8.8)
  • Add 30.3 g Tris to 150 mL water
  • Adjust pH 8.8 with HCL
Bring to 250 mL with water.
Stacking Gel Buffer (0.375M Tris HCl, pH 6.8)
  • Add 11.4 g Tris to about 150 mL water
  • Adjust to pH 6.8 with HCl
Bring to 250 mL with water.
Catalyst-Ammonium Persulfate (Make fresh the day of use)
  • 100 mg Ammonium Persulfate
Bring to 2 mL in water.
10% SDS
  • 10.0 g SDS
Bring to 100 mL with water.
50% Sucrose
  • 50.0 g Sucrose
Bring to 100 mL with water.
   

Separating gel

The following recipes are for approximately 25 mL of separating gel, enough for four 1.0-mm thick mini gels. Scale volumes proportionally based on the number of gels to be cast.

Solution 6% Gel 8% Gel 10% Gel 12% Gel 14% Gel 16% Gel 18% Gel 20% Gel
50% Acrylamide/BIS 3.0 mL 4.0 mL 5.0 mL 6.0 mL 7.0 mL 8.0 mL 9.0 mL 10.0 mL
Separating Gel Buffer 9.4 mL 9.4 mL 9.4 mL 9.4 mL 9.4 mL 9.4 mL 9.4 mL 9.4 mL
10% SDS 250 µL 250 µL 250 µL 250 µL 250 µL 250 µL 250 µL 250 µL
50% Sucrose* 4.0 mL 4.0 mL 4.0 mL 4.0 mL 4.0 mL 4.0 mL 4.0 mL 4.0 mL
Water 7.8 mL 6.8 mL 5.8 mL 4.8 mL 3.7 mL 2.7 mL 1.7 mL 750 µL
TEMED** 6.25 µL 6.25 µL 6.25 µL 6.25 µL 6.25 µL 6.25 µL 6.25 µL 6.25 µL
Catalyst** 625 µL 625 µL 625 µL 625 µL 625 µL 625 µL 625 µL 625 µL

*Optional but recommended because it makes it easy to form a good interface between the separating gel and the overlay. If omitted, increase the amount of water added to make up for the volume of the sucrose solution (increase the water by 4.0 mL for the above tables).
**Add these last and mix well just before the gel is to be poured.

Stacking gel

Following recipe is for 4% Stacking Gel (12.5 mL)

Solution 4%
50% Acrylamide/BIS 1.0 mL
Stacking Gel Buffer 4.2 mL
10% SDS 125 µL
Water 6.3 mL
TEMED* 5.0 µL
Catalyst* 1.0 mL

*Add these last and mix well just before the gel is to be poured.