Westem hybridization, also known as western blotting, is a method for the detection of specific proteins that integrate protein electrophoresis, blotting, and immunoassay by transferring the protein components separated by SDS-PAGE to an immobilized matrix, and then analyzing the presence and content of the target protein with specific antibodies by immunological methods.
The principle is to extract the total protein or target protein from biological cells, dissolve the protein sample in a solution containing detergent and reducing agent, separate the protein by SDS-PAGE electrophoresis according to the molecular mass, and then blot the separated protein strips onto the solid phase membrane (nitrocellulose membrane or nylon membrane), and incubate the membrane with a high concentration of protein solution to seal its non-specific sites, and then add specific resistant bodies (primary antibodies), and the target protein (antigen) on the membrane binds to the primary antibody. Then add a labeled secondary antibody that can specifically bind to the primary antibody (usually when the primary antibody uses rabbit** antibody, the secondary antibody is commonly used sheep anti-rabbit immunoglobulin antibody), and finally detected by a specific reaction with a labeled compound (generally horseradish peroxidase or alkaline phosphatase) on the secondary antibody. According to the test results, the expression of the target protein, the expression amount and molecular weight in the cells of the tested organisms can be known.
A typical western hybridization consists of the following four steps: protein extraction and gel preparation electrophoresis. Extract the total protein or target protein according to the conventional method, prepare the SDS-PAGE gel for Western blot analysis, peel off the gel after electrophoresis, remove the gel, and rinse or equilibrate the gel with electroporation; Transfer. Select a suitable membrane and perform methanol treatment and distilled water rinsing, and electrotransfer of proteins; Blocking and hybridization. After the transfer is completed, the membrane is taken out to rinse and sealed, combined with primary antibody, washed, bound with secondary antibody, washed; Luminescence or chromogenic identification. Alkaline phosphatase AP-NBT BICP chromogenic method or horseradish peroxidase HRP-ECL luminescence method is generally used.
Dyeing principle:
Ponceau red, which is negatively charged, can bind to positively charged amino acid residues, while Ponceau can also bind to the non-polar regions of the protein, resulting in red bands. It can be used for the detection of proteins on PVDF membranes, nitrocellulose membranes and cellulose acetate membranes, but not for the detection of proteins on nylon membranes.
How to use:
Immerse PVDF membrane, nitrocellulose membrane or cellulose acetate membrane in Ponceau red staining solution, shake for 5-10 minutes or longer, remove the blotting membrane, rinse 2-3 times with distilled water, PBS or other appropriate solutions, clear bands can appear, and the results are recorded; Rinse 2-3 times with distilled water, PBS, or other appropriate solutions for 3-5 minutes each time to remove ponceau red for subsequent Western blot testing.