Western blotting uses specific antibodies to identify proteins that have been separated based on size by gel electrophoresis. The immunoassay uses a membrane made of nitrocellulose or PVDF (polyvinylidene fluoride). The gel is placed next to the membrane and application of an electrical current induces the proteins to migrate from the gel to the membrane. The membrane can then be further processed with antibodies specific for the target of interest, and visualized using secondary antibodies and detection reagents.

Solutions and reagents

Sample preparation

Loading and running the gel

Transferring the protein

Antibody staining

10% SDS

For 100 mL solution:

10 g SDS

Add distilled water to a final volume of 100 mL at 50℃ and store at room temperature.

10% AP (ammonium persulfate)

For 1 mL solution:

0.1 g AP

Dissolve in 1 mL distilled water and store at 4℃.

1.5 M Tris-HCl (pH8.8)

1.5 M Tris

For 100 mL solution:

18.17 g Tris base (MW: 121.14 g)

Add distilled water to a final volume of 100 mL, check the pH and adjust to 8.8.

0.5 M Tris-HCl (pH6.8)

0.5 M Tris

For 100 mL solution:

6.06 g Tris base (MW: 121.14 g)

Add distilled water to a final volume of 100 mL, check the pH and adjust to 6.8.

30%(w/v) Acrylamide Solution

29 g

1 g

Add distilled water to a final volume of 100 mL, check the pH (≤7.0) and store in brown bottles.

​Laemmli 2X buffer/loading buffer

4% SDS

10% 2-mercaptoethanol

20% glycerol

0.004% bromophenol blue

125 mM Tris-HCl

Check the pH and adjust to 6.8.

​Running buffer (Tris-Glycine/SDS)

25 mM Tris base

190 mM glycine

0.1% SDS

For 1 L 5x storage buffer:

15.14 g Tris base (MW: 121.14 g)

71.32 g Glycine (MW: 75.07 g)

5 g SDS

Add distilled water to a final volume of 1000 mL.

For a 1x solution, mix 1 part of the 5x storage buffer with 4 parts distilled water and adjust pH to 8.3.

​Transfer buffer (wet)

25 mM Tris base

190 mM glycine

20% methanol

0.1% SDS (for proteins larger than 80 kDa)

For 1 L buffer:

3.03 g Tris base (MW: 121.14 g)

14.26 g Glycine (MW: 75.07 g)

Methanol 200 mL

1 g SDS (for proteins larger than 80 kDa)

Add distilled water to a final volume of 1000 mL, check the pH and adjust to 8.3.

​Transfer buffer (semi-dry)

48 mM Tris base

39 mM glycine

20% methanol

0.04% SDS

For 1 L buffer:

5.81 g Tris base (MW: 121.14 g)

2.93 g Glycine (MW: 75.07 g)

Methanol 200 mL

0.4 g SDS

Add distilled water to a final volume of 1000 mL.

​Blocking buffer

3–5% milk or BSA (bovine serum albumin)

Add to TBST buffer.

Mix well and filter.

Failure to filter can lead to spotting, where tiny dark grains will contaminate the blot during color development.

​Sample preparation

1. Remove a small volume of lysate to perform a protein quantification assay. Determine the protein concentration for each cell lysate.

2. Determine how much protein to load and add an equal volume 2X Laemmli sample buffer.​

3. To reduce and denature your samples, boil each cell lysate in sample buffer at 100℃ for 5 min. Lysates can be aliquoted and stored at -20℃ for future use.

​​Loading and running the gel

1. A reducing gel should be used unless non-reducing conditions are recommended on the antibody datasheet.

The gel percentage required is dependent on the size of your protein of interest:

Gradient gels can also be used.

2. Load equal amounts of protein into the wells of the SDS-PAGE gel, along with molecular weight marker. Load 20–30 μg of total protein from cell lysate or tissue homogenate, or 10–100 ng of purified protein.

3. Run the gel for 1–2 h at 100 V. The time and voltage may require optimization, following the manufacturer's instructions.

​Transferring the protein from the gel to the membrane

1. The membrane can be either nitrocellulose or PVDF. Activate PVDF with methanol for 10 min and rinse with transfer buffer before preparing the stack. Prepare the stack as follows:

Anode(+)-Sponge-Filter paper-Membrane-Gel-Filter paper-Sponge-Cathode(-)

2. For wet transferring, the buffer need to be precooled and tank should be put on ice for 1 h under 300 mA (or 100 V), or overnight for 30 V.

For hemi-dry transferring, using 0.8 mA per cm2 membrane for 1.5 h.

The time and voltage of transfer may require some optimization, following the manufacturer's instructions. Transfer of proteins to the membrane can be checked using Ponceau S staining before the blocking step.

​Antibody staining

1. Block the membrane for 30 min to 1 h at room temperature or overnight at 4℃ using blocking buffer.

2. Incubate the membrane with appropriate dilutions of primary antibody in blocking buffer for 1 h at 37°C, or overnight at 4℃; other conditions (including proper dilution rate of the antibody) can be optimized according to the antibody datasheet.

3. Wash the membrane in three washes of TBST, 5-10 min each.

4. Incubate the membrane with the recommended dilution of conjugated secondary antibody in blocking buffer at room temperature for 1 h.

5. Wash the membrane in three washes of TBST, 5-10 min each.

6. For signal development, follow the kit manufacturer's recommendations. Remove excess reagent and cover the membrane in transparent plastic wrap.

7. Acquire image using darkroom development techniques for chemiluminescence, or normal image scanning methods for colorimetric detection.