How To Prepare Samples For Western Blot Analysis
Western immunoblotting or blotting is an indispensable technique, almost every published document in the field of molecular cell biology uses Western blotting to detect specific proteins in samples of tissue homogenates or 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 western blot service price via https://www.bosterbio.com/services/assay-services/western-blotting-service. 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.
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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 bovine serum albumin, skim milk, 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 horseradish peroxidase or a fluorophore.
How do you prepare your samples?
- Choose the Right Lysis Buffer for Your Sample
- Whether to add detergents or not?
- Add Protease and Phosphatase Inhibitors
- Determine Protein Concentration before you load your samples
- Load the Proteins