![]() ![]() Overlay the stacking gel with the separating gel, after removing the water.Wait for 20-30 minutes until the gel turning solidified. First add stacking gel solution carefully until the level is equal to the red bar holding the glass plates Figure 2.After preparing in Table 1 the 10% stacking gel solution and separating gel solution, assemble the rack for gel solidification Figure 1.Heat the samples with dry plate for 5 minutes at 100✬.(Tip: Total volume of 15 μL per lane is suggested). Add 5 μL sample buffer to the sample, and make the volume in each lane equalized using dd H2O.Determine the volume of protein extract to ensure 50μg in each well (concentration=mass/volume).Measure the concentration of protein using a spectrophotometer.Transfer supernatant to a fresh tube and store on ice or frozen at -20☌ or -80☌.Incubate for 30 minutes on ice, and then spinning for 10 minutes at 12,000 RPM, at 4☌.(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 5-10 minutes and discard the supernatant.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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