Hello Gaurav,

The detailed protocol for Western Blot may be found in various molecular biology, biochemistry, and cell biology textbooks. These texts often include detailed steps for gel electrophoresis, membrane transfer, blocking, antibody incubation, and analysis. Look for sections on protein analysis techniques. Nevertheless, I have provided a brief protocol but given a detailed description on the staining procedure for DAB (step 5).

The protocol is as follows:

1. Sample Preparation

a. Prepare your protein samples by solubilizing, reducing and denaturing.

b. Load samples onto the gel (details of SDS-PAGE preparation may be found in the textbook) using a known amount of protein (e.g., 10-50 μg per lane) into the wells of an SDS-PAGE gel.

c. Run the gel to separate proteins based on size using electrophoresis.

2. Protein Transfer

a. Prepare transfer buffer namely, Tris-Glycine transfer buffer consisting of 25 mM Tris, 192 mM glycine, and 20% methanol (vol/vol), with a pH of 8.3. Sometimes, a small amount of SDS (0.1-0.25%) is also added.

b. Assemble the transfer sandwich by placing the gel, membrane, and filter papers in the correct order within the transfer apparatus, details of which can be obtained from the textbook.

c. Apply an electric field to transfer proteins from the gel to the membrane.

d. Check the transfer efficiency by staining the membrane with Ponceau S to visualize the protein bands.

3. Blocking

Incubate the membrane in blocking buffer (5% non-fat dry milk or BSA in TBST) with gentle shaking for a specific time (e.g., 1 hour at room temperature or overnight at 4°C).

4. Antibody Incubation

a. Incubate the membrane with diluted primary antibody (diluted in blocking buffer according to the manufacturer's recommendations), usually overnight at 4°C with gentle shaking.

b. Rinse the membrane with TBST to remove unbound primary antibody.

c. Incubate the membrane with the diluted secondary antibody (diluted in blocking buffer) for a specific time (e.g., 1 hour).

d. Wash the membrane thoroughly with TBST to remove unbound secondary antibody.

5. Protein Detection with DAB

a. Prepare Tris buffered saline (20 mM Tris, 500 mM NaCl, pH 7.5), 2Litres: Dissolve 4.84 g Tris base and 58.48 g NaCl in approximately 1.5Litres distilled, deionized H2O. Adjust to pH 7.5 with HCl. Adjust the volume to 2Litres with distilled, deionized H2O.

b. Prepare DAB color development solution: Dissolve 50 mg DAB in 100 ml TBS. Add 10 µl 30% H2O2. Make this solution just before use.

c. Immerse the membrane in DAB color development solution and incubate for 10-20 minutes. DAB is used for detecting antigens bound to the membrane. This substrate develops a brown, insoluble product on the membrane surface after exposure to horseradish peroxidase conjugated antibodies. Protein will become visible immediately as brown bands or dots. Lesser concentrations may take as long as 15-20 minutes. Do not overdevelop, as high backgrounds may result. Please note that while DAB might be sufficient for detecting abundant proteins, lower abundance proteins are more effectively detected using chemiluminescence or other sensitive methods.

d. Stop the color development by immersing the membrane in distilled H2O for 10 minutes. Change the water at least once to remove remaining color development solution. Examine the membrane for brown bands and document your results.

Best,

@Malcolm Nobre thanks.

Hello Gaurav,

No, you cannot. To detect housekeeping proteins on the same blot, you will have to use a different detection method, such as chemiluminescence or fluorescence, that allows for stripping and reprobing. Alternatively, you can cut the blot into strips, with each strip used for a different protein target.

If you are developing the blot using DAB for detection, it cannot be stripped and reprobed for housekeeping proteins because DAB is a chromogenic substrate, producing a visible precipitate at the reaction site, which is difficult or impossible to remove without damaging the membrane. Unlike chemiluminescent or fluorescent detection methods, the DAB precipitate remains on the membrane even after the reaction is stopped, making it unsuitable for stripping and reprobing.