Transfer the mixture into micro centrifuge tubes via Pipette. Add PBS tissue culture flask or dish and use a cell scraper to dislodge the cells.(Tip: Keep tissue culture dish on ice throughout). Washing cells in the tissue culture flask or dish by adding cold phosphate buffered saline (PBS) and shaking gently.Below is the protocol to extract proteins from adherent cells. Protein can be extracted from different kind of samples, such as tissue or cells. This paper will first describe the protocol in detail for western blot, accompanied by theory to rationalize the protocol and followed by the theoretical explanation of the procedure, and in the later section, troubleshooting tips for common problems. The thickness of the band corresponds to the amount of protein present in the sample thus doing a standard can indicate the amount of protein present. As the antibodies specifically only bind to the target protein, only one band should be visible. The bound antibodies are then detected by developing the film. The unbound antibody from the membrane is washed off leaving only the bound antibody to the protein of interest. The membrane is then incubated with labels primary and secondary antibodies specific to the protein of interest. The results on gel are then transferred to a membrane producing a band for each protein. This technique involve separation of mixture of proteins based on molecular weight, and thus by type, through gel electrophoresis. Western blot is used in molecular and biochemical research to separate and identify the proteins of interest.
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