Western Blot Protocol

Western Blot (WB) refers to the electrophoretic transfer of proteins from sodium dodecyl sulfate polyacrylamide gels to sheets of polyvinylidene difluoride (PVDF) or nitrocellulose (NC) membrane, followed by immunodetection of proteins using antibodies with fluorescent or chemiluminescent detection.

General Steps

• Sample Preparation
• SDS-PAGE Gel Electrophoresis
• Protein Transfer
• Immunoblotting
• Detection

Buffer and Reagents

• Lysis Buffer Recipes
1. NP-40 Buffer
• 150 mM NaCl
• 1.0% NP-40 or Triton X-100
• 50 mM Tris pH 8.0
2. RIPA Buffer
• 150 mM NaCl
• 1.0% NP-40 or Triton X-100
• 0.5% sodium deoxycholate
• 0.1% SDS
• 50 mM Tris, pH 8.0
3. Tris-HCl Buffer
• 20 mM Tris-HCl, pH 7.5
4. Tris-Triton Buffer
• 10 mM Tris, pH 7.4
• 100 mM NaCl
• 1 mM EDTA
• 1 mM EGTA
• 1% Triton X-100
• 10% glycerol
• 0.1% SDS
• 0.5% deoxycholate
• Blocking Buffer
1. 1X TBST
2. 5% non-fat dry milk OR 5% BSA
• Loading Buffer Recipes

2X Laemmli Buffer Recipe

1. 4% SDS
2. 10% 2-mercaptoethanol
3. 20% glycerol
4. 0.004% bromophenol blue
5. 0.125 M Tris HCl
• Running Buffer Recipes
1. 25 mM Tris base
2. 192 mM glycine
3. 0.1% SDS

Adjust pH to 8.3

• Transfer Buffer Recipes

1X Transfer Buffer (Wet)

1. 25 mM Tris base
2. 192 mM glycine
3. 20 % methanol

Adjust pH to 8.3

1X Transfer Buffer (Semi-Dry)

1. 48 mM Tris base
2. 39 mM glycine
3. 20 % methanol

Adjust pH to 8.3

Methods

1. Refer to Preparation of Lysate from Cell Culture or Preparation of Lysate from Tissues for detail lysate preparation steps.
2. Determine the protein concentration of each cell lysate and how much protein to load. Add an equal volume 2X Laemmli buffer.

Note: Recommended loading amount: 10-50 μg/lane.

3. Reduce and denature the samples by boiling the lysates in sample buffer at 95-100˚C for 5 minutes.

Note: This step should be skipped if the antibody datasheet recommends non-reducing or non-denaturing conditions.

4. Prepare or purchase a pre-made gel of appropriate polyacrylamide percentage to best resolve your protein of interest based on molecular weight (MW).

Note: If the MWs of your targets range from low to high MW sizes or if you plan to probe a blot for multiple proteins varying in size, gradient gels may be necessary to achieve efficient separation of proteins.

5. Load samples containing equal amounts of protein prepared in sample buffer into SDS-PAGE wells. Protein marker should be loaded in one of the lanes.
6. Fill the electrophoresis apparatus with 1X running buffer.
7. Run the gel.

Note: Voltage may vary depending on research needs. 1 hour at 100V is a standard guideline.

8. Prepare the polyvinylidene difluoride (PVDF) membrane: wetting it in methanol for 30 seconds and then soaking it briefly in distilled water followed by 1X transfer buffer.

Note: Handle the membrane carefully, ideally with rounded tweezers to avoid scratching or puncturing the surface.

9. Soak filter papers and sponges in the transfer buffer for 5-10 mins prior to assembly of the transfer sandwich.
10. Once electrophoresis is complete, remove the gel from the electrophoresis apparatus and equilibrate it by soaking it in transfer buffer for 5-10 mins.
11. Prepare the sandwich according to the illustration below. Sequentially assemble the layers of the sandwich. Gently remove any air bubbles with pipette.

Note: Bubbles between the gel and the membrane will inhibit the transfer of proteins to the membrane.

12. Place the sandwich into a transfer cassette and perform semi-dry or wet transfer according to the manufacturer’s instructions.

There are also individual instructions for Sample Preparation, SDS-PAGE Gel Electrophoresis, Membrane Transfer steps. Refer to corresponding section for more detail.

Immunoblotting and Detection

13. After transfer, briefly wash the membrane in 1X TBST.

Note: Coomassie staining of the gel can help assure that proteins were completely transferred from the gel to the membrane.

14. Incubate membrane in blocking solution for 1 h at RT or overnight at 4˚C.
15. Dilute the primary antibody to working concentration in 1X TBST with blocking agent. Incubate the membrane in primary antibody solution for 1 h at RT or overnight at 4˚C with gentle rocking.
16. Wash the membrane with 1X TBST three times for 5-10 minutes each with gentle rocking.
17. Incubate the membrane in secondary antibody solution for 1 h at RT with gentle rocking.
18. Wash the membrane in 1X TBST three times for 5-10 minutes each with gentle rocking.
19. Incubate the membrane in the substrate according to manufacturer’s directions.

Note: Typical incubation times are 1-5 minutes.

20. Carefully remove the membrane from the detection reagent. Expose the membrane to autoradiography film in a dark room or image with a chemiluminescent imaging system.

Note: Following target protein detection, primary antibody and secondary antibody can be used to reprobe the same blot for a second protein. To strip and reprobe your blot, please read our Stripping and Reprobing Protocol.

View our Western Blot Troubleshooting to solve your problem.