Immunoblotting, also known as western blot, is a method of detecting a certain protein in a complex sample based on the specific binding of antigen and antibody.Western blot is a new immunobiochemical technology developed on the basis of gel electrophoresis and solid-phase immunoassay technology.Because of the high resolution of SDS-PAGE and the high specificity and sensitivity of solid-phase immunoassay, western blot has become a routine technique for protein analysis.Western blot is often used to identify a certain protein, and can perform qualitative and semi-quantitative analysis of the protein
- tProtein extractiont
- ttThe experimental object is a tissue sample. Take an appropriate amount (250~500mg) of a fresh tissue sample or a properly preserved tissue sample, add 1ml of total protein extraction reagent (or nucleoprotein extraction reagent) containing protease inhibitors, and extract the total after homogenizationProtein (or nuclear protein).tt tt
- ttThe test object is a cell sample(Cell culture), Take 1×106～1×107 cells for each sample, wash the cells with PBS, add 0.1ml~1ml total protein extraction reagent (or nuclear protein extraction reagent) containing protease inhibitor to extract total protein (or nuclearprotein).tt t
2. Protein quantification: Follow the instructions of the BCA protein quantification kit to determine the sample concentration.
3. Denaturing polyacrylamide discontinuous gel electrophoresis(SDS-PAGE): Load the prepared sample solution and pre-stained protein marker separately, add the standard to the first well, and separate the protein by electrophoresis.
4. The protein is transferred to the PVDF membrane, and the filter paper gel cellulose interlayer is assembled according to the instructions of the Bio-Rad protein transfer device. Under the condition of 30mA constant current, transfer overnight at 4°C.
5. Blocking of western blot membrane and antibody incubation
- tThe membrane was incubated in a 5% skimmed milk powder solution at room temperature for 1 hour to block non-specific binding on the membrane.t t
- tThe blocked membrane is added with primary antibody and incubated for 1.5 hours at room temperature to bind the antigen and antibody.t t
- tAdd HRP-labeled secondary antibody to bind the primary antibody, and incubate the membrane for 1 hour at room temperature.Adding HRP-labeled GAPDH antibody can simultaneously detect GAPDH content.t
6. Western blot detection: detection by chemiluminescence method, the membrane is incubated with the chemiluminescence substrate, and exposed and developed by X film.The scanned image is saved as a computer file, and the gray value of each specific band on the image is digitized with GIS1000 analysis software.
7. Western blot data analysis: the gray value of the target protein is divided by the gray value of the internal reference GAPDF to correct the error, and the result represents the relative content of the target protein of a sample.
8. Provide experimental report, including detailed experimental methods and relevant data of western blot experiment results.
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