![]() (Tip: If protein concentration is not high enough at the end, it is advised to repeat the procedure with a higher proportion of protease inhibitor cocktail). Add 180 μL of ice cold cell lysis buffer with 20 μL fresh protease inhibitor cocktail.Centrifuge at 1500 RPM for 5 minutes and discard the supernatant.Pipette the mixture into microcentrifuge tubes. Add PBS and use a cell scraper to dislodge the cells.(Tip: Keep tissue culture dish on ice throughout). Wash cells in the tissue culture flask or dish by adding cold phosphate buffered saline (PBS) and rocking 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 will be followed by the theoretical explanation of the procedure, and in the later section, troubleshooting tips for common problems. The paper will first describe the protocol for western blot, accompanied by pictures to help the reader and theory to rationalize the protocol. The thickness of the band corresponds to the amount of protein present thus doing a standard can indicate the amount of protein present. As the antibodies only bind to the protein of interest, only one band should be visible. The bound antibodies are then detected by developing the film. The unbound antibody is washed off leaving only the bound antibody to the protein of interest. The membrane is then incubated with labels antibodies specific to the protein of interest. These results are then transferred to a membrane producing a band for each protein. In this technique a mixture of proteins is separated based on molecular weight, and thus by type, through gel electrophoresis. Western blot is often used in research to separate and identify proteins. Western blot: Technique, theory, and trouble shooting. How to cite this URL: Mahmood T, Yang PC. How to cite this article: Mahmood T, Yang PC. Keywords: Bio-medical research, protein, western blot This paper will attempt to explain the technique and theory behind western blot, and offer some ways to troubleshoot. The technique uses three elements to accomplish this task: (1) separation by size, (2) transfer to a solid support, and (3) marking target protein using a proper primary and secondary antibody to visualize. By using a western blot, researchers are able to identify specific proteins from a complex mixture of proteins extracted from cells. Western blotting is an important technique used in cell and molecular biology.
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