How To Prepare Samples For Western Blot Analysis

Western immunoblotting, or blotting, is an essential technique. Nearly every published paper in molecular biology uses Western blotting for the detection of specific proteins in tissue homogenates and cell lysates.

Western blotting combines polyacrylamide gel electrophoresis (PAGE) or SDS-PAGE resolution and antibody specificity for detecting target proteins. If you are thinking to hire wester blot services then you can check the western blot service price via www.bosterbio.com/services/assay-services/western-blotting-service

Western Blotting - MyBioSource Learning Center

Image Source: Google

Proteins are separated by molecular weight in SDS-PAGE and transferred from a polyacrylamide gel to a membrane (nitrocellulose or PVDF), creating an exact copy of the protein separation pattern on the membrane. 

After the protein is transferred to the membrane, the membrane must be “blocked” to “block” the non-specific binding sites on the membrane surface. The blockade is usually performed with skim milk, bovine serum albumin, or whole casein milk.

Without blocking, the antibody binds to a non-specific membrane binding site and makes detection of the target protein more difficult, leading to false-positive results. Once the proteins are blocked on the binding membrane after being blocked, they can be assayed with a specific primary antibody for the protein of interest. 

Absorbed or bound proteins can be detected with a primary antibody bound to the reporter molecule, such as a fluorophore or horseradish peroxidase.   

How do you prepare your samples?

1. Select the Most Appropriate Lysis Buffer for Your Sample

2. Should I use detergents or not?

3. Include Protease and Phosphatase Inhibitors in the mix.

4. Before loading your samples, determine the protein concentration.

5. Prepare the Proteins

Continue Reading