Question

What is Western blot used for? Describe how you would perform a Western blot, starting from the sample preparation step to the final analysis.

Answer 1

Western blot is often used to detect, separate, and identify specific protein molecules from among a mixture of proteins. It is also called immunoblotting and is used in qualitative and semi-quantitative analyses of proteins.

Steps in western blotting

1. Sample preparation

First step in sample preparation is Lysis of cells. Different methods are used to separate proteins from biological materials such as Mechanical disruption, Liquid homogenization, Sonication, Freeze/ thaw and Grinding with liquid nitrogen. Second step in sample preparation is Protein quantification. This can be done in many ways as Absorbance method, Biuret method, Lowry method and BSA. Ready-to-use reagents and extraction buffers containing detergents are used for extraction of soluble proteins. After centrifugation with the reagents and buffers, cleared supernatant (cell extract) is used to proceed with analysis.

2. Gel electrophoresis.

The protein samples are loaded onto a gel (SDS PAGE) for protein separation. Proteins are separated based on their size. A commercially pre-cast gel or self-prepared PAGE gel could be used. To enable access of the antibody to the epitopes, which reside within the 3D conformation of the protein, it is necessary to unfold the protein. The denaturing is done by loading buffer with the anionic denaturing detergent sodium dodecyl sulphate (SDS) and boiling the mixture at 95-100°C for 5 minutes. A loading dye is added to the sample to monitor the protein migration. When gel is placed in the electrophoretic tank with the running buffer that will conduct current, the negatively charged proteins migrate away from the anode. After separation, the gel is stained with Coomassie blue to ensure the proteins have migrated evenly and uniformly.

3. Gel transfer

The gel and membrane are placed between filter paper and sponge pads (wet transfer) to transfer the separated proteins in an electrical field from the gel onto a membrane. Membrane can be PVDF or nitrocellulose. The sandwich ((sponge/paper/gel/membrane/paper/sponge)) is submerged in transfer buffer during the transfer. Western Blot Stain are used to ensure the protein transfer.

4. Membrane blocking

After transfer, the membrane is removed and incubated in a container with gentle agitation with a blocking buffer to prevent non-specific background binding. Non-fat milk or BSA are used as a blocking solution.

5. Incubation with antibodies

The blocked membrane is incubated with primary antibodies that are specific for the epitope of the target protein. After treating with blocking agents and washing with washing buffer for many times, the membrane is incubated with secondary antibodies with conjugates. Again, it is treated with blocking agents and washing buffer.

6. Detection

The conjugate in the secondary antibodies are used to visualize the target protein. Secondary antibodies can be conjugated with different molecules to make the visualization and quantification possible in different ways. For example, for HRP conjugating secondary antibodies, ECL is traditionally used for visualization. Colorimetric assays can be used for quantification of antibodies conjugated with enzymes that produce color and light. Fluorescent can be used which are detected with digital imager.