Overview

Western blotting is a common molecular biology technique to identify specific proteins and determine relative mass.

Principle

This technique uses the electrophoretic mobility of proteins and the specificity of the antibody-antigen interaction. The first step in western blotting is to separate the proteins in a sample using gel electrophoresis (Sodium dodecyl-sulfate polyacrylamide gel electrophoresis or SDS-PAGE). The separated proteins are transferred out of the gel and onto a solid support matrix (e.g. nitrocellulose or PVDF membrane). Then we can use antibodies specific to the target protein to visualize the protein of interest.

Application

1. Detecting protein expression:

Identify specific proteins and determine the relative abundance of a protein target.

2. Analysis of protein-protein interactions:

Study protein interactions through the western blot analysis following co-immunoprecipitation (Co-IP).

3. Detecting post-translational modifications:

Use specific antibodies to detect post-translation modifications (PTMs) such as phosphorylation, acetylation, etc.

4. Analysis of subtypes from proteins:

Differentiate different subtypes of proteins using different antibodies.

Advantages

1. Specificity: Detect the protein of interest using antibodies specific to the target protein.

2. Sensitivity: Detect trace amount of proteins in samples through signal amplification.

3. Reliable quantification: Conduct semi-quantitative analysis with standard and control samples.

Limitations

1. Sample preparation: High-quality samples and appropriate sample preparation steps are necessary.

2. Antibody quality: The selection and quality of antibodies can make or break results.

3. Background signal: Non-specific binding may lead to high background signals.

Procedures

1. Sample preparation

Ensure a typical starting point per well before different interventions to cells. Use the RWD C100-Pro automated cell counter for cell counting and plating.

2. Gel electrophoresis

Insert a 10% gel into the electrophoresis chamber (on the left), and load samples to the wells in predetermined amounts (in the middle). Run the gel at 80V and switch to 120V when proteins migrate through the top of the gel until the position indicated on the right.

3. Protein transfer onto a membrane

Next transfer proteins from the gel to a PVDF membrane. Prepare for transfer by obtaining transfer buffer, methanol (for PVDF membrane activation), PVDF membrane, transfer clamp, and membrane roll. Wash the gel plate with ddH2O, remove the electrophoresis buffer, and place it in the transfer buffer. Place the gel and membrane in a transfer sandwich tightly clamped with the gel on the black part and the activated PVDF membrane on the white part as seen below.

Place the clamp in the transfer tank. Black to black and white to red. Don’t go the other way around, or your efforts are ruined. Then hook up the power supply the same way. Red to red and black to black.

A rapid protocol can transfer proteins of 42 kD to membrane in 25 minutes at 400 mA.

4. Blocking

After transfer, choose whether to cut the membrane based on your needs. You can block the membrane using either commercially available blocking buffers, 5% BSA, or 5% non-fat dried milk. If you need to cut the membrane, be sure not to let the membrane dry and keep it moist. Block the membrane at room temperature on a shaker for an hour (Figure 7).

5. Antibody incubation

Antibody incubation comes after membrane blocking. Incubate primary antibody overnight at 4°C. Refer to the manual for the optimum primary antibody concentration for western blotting. Consider the host species of the primary antibody, i.e. whether it is produced in mice or rabbits so that an anti-species secondary antibody is produced for binding on the following day. Wash the membrane three times with TBST, 5-10 min each, for secondary antibody incubation. The secondary antibody should be prepared at the concentration as instructed and incubated for an hour at room temperature before exposure.

6. Imaging and analysis

After incubation, run imaging analysis with a western blot imager. RWD’s qTouch western blot imager is used in the experiment. It uses a highly sensitive photosensitive chip for contact imaging to capture western blot signals without loss, ensuring clear imaging. Simply place the protein membrane on the chip and run the software for image capture and grayscale analysis. It is highly recommended for everyone to try it.