Western Blot – 4. Staining

You can skip staining step. However, if you want to know that proteins were transferred from the gel to membrane, you can stain either the gel or the membrane.

Membrane Staining

Ponceau S Staining

Ponceau S staining is the method used for evaluating the transfer efficiency of western blot. It can be used for PVDF and nitrocellulose membranes. Ponceau S binds not only the positively charged amino groups but also the non-polar regions of proteins. Therefore, Ponceau s stain is not suitable for nylon membranes as nylon is positively charged allowing dyes to bind to membrane strongly.

Procedure

1. Incubate the membrane in Ponceau S staining solution for 5 min on shaker at RT.

2. Remove Ponceau S solution. Note. It can be re-used until the stained band strength is noticeably lower. Keep the used solution in 50 ml tube covered with foil.

3. Wash with H2O for 5 min on shaker at RT, 3 times. Note. You can take a photo of the membrane in this step for your record.

4. After confirming the protein bands, wash the membrane with TBS-T or PBS-T for 10 min on shaker at RT, 3 times. Note. Even if the bands were not de-stained perfectly, you can move to the next step. The bands will be de-stained during the blocking step.

Gel staining

Coomassie Blue Staining

There are two forms of dye for Coomassie staining. One is G-250 and the other one is R-250. Both dyes offer similar sensitivity but G-250 is more sensitive than R-250. However, R-250 is cheaper and it provides better resolution. Coomassie Blue stained gel is not recommended for western blot as the transfer will be fairly inefficient due to methanol/acetic acid solution.

Coomassie Blue G-250 Staining

1. Soak the gel in 0.5% Coomassie Blue G-250 (in 50% methanol/ 10% acetic acid solution) and gently shake for 5 min.

2. Rinse the stained gel in H2O, 2‐3 times for 5 minutes each.

3. Soak the gel in 40% HPLC grade methanol/10% acetic acid solution and gently shake for 5 min, 3 times.

4. Rinse the stained gel in H2O until bands are very clean.

5. The gel can be stored in 1% acetic acid or H2O at 4°C.

Coomassie Blue R-250 Staining

1. Soak the gel in 50% HPLC grade methanol/ 10% acetic acid solution for 1 h to overnight.

2. Stain the gel in the above solution with 0.5% Coomassie Blue R-250 for 20 min.

3. De-stain the gel in 50% HPLC grade methanol/ 10% acetic acid solution for 2 h. Change the solvent at least twice during de-staining. De-stain until background is clear.

4. The gel can be stored in 1% acetic acid or H2O at 4°C.

Silver Staining

Silver staining is the most sensitive colorimetric way for detecting total protein. If formaldehyde or glutaraldehyde was used during fixing step, the stained gel can’t be used for mass spectrometry. Below protocol is optimized for mass spectrometry.

Fixation

1. Incubate the gel with Fixing solution (40% ethanol, 10% acetic acid, 50% H2O) for 1 h.

2. Wash the gel with H2O for at least 30 min. Note. Overnight washing with several changes of water will remove all acetic acid, reduce background staining, and increase sensitivity.

Sensitizing

1. Incubate the gel with 0.02% sodium thiosulfate (0.04 g Na2S2O3, 200 ml H2O) for 1 min. Note. Longer incubation will decrease peptide recovery from the gel.

2. Wash the gel in H2) for 20 sec. 3 times.

Silver reaction

1. Incubate the gel for 20 min with cold 0.1% silver nitrate solution (0.2 g AgNO3, 200 ml H2O, 0.02% formaldehyde (add 40 ul 35% formaldehyde just before use)). Note. Staining is enhanced with cold silver nitrate solution.

2. Wash the gel with H2O for 20 sec. 3 times.

3. Place the gel in a new staining tray. Note. Residual AgNO3 on the gel surface and staining tray will increase background staining.

4. Wash the gel with H2O for 1 min.

Developing

1. Incubate with 3% sodium carbonate (7.5 g Na2CO3 in 240 ml H2O), 0.05% formaldehyde (add 125 ul 35% formaldehyde just before use) until desired intensity of staining occurs. Note. Change the solution when it turns yellow.

2. Wash the gel with H2O for 20 sec.

Stopping

1. Terminate developing with 5% acetic acid for 5 min.

2. Wash the gel with water for 5 min.

Storage

1. Leave the gel in 1% acetic acid solution at 4C. Wash the gel with H2O for 10 min, 3 times before MS analysis

Mortz E, Krogh TN, Vorum H, Görg A. Improved silver staining protocols for high sensitivity protein identification using matrix-assisted laser desorption/ionization-time of flight analysis. Proteomics. 2001 Nov;1(11):1359-63. doi: 10.1002/1615-9861(200111)1:11<1359::AID-PROT1359>3.0.CO;2-Q. PMID: 11922595.