Encyclopedia of Experiments: Biological Techniques
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Transcript
To perform the western blot, add 20 microliters of 4X loading buffer to the beads, and heat at 95 degrees Celsius for 10 minutes. Simultaneously, heat the lysate controls at 95 degrees Celsius for 5 minutes. Load the lysates, immunoprecipitants, and a protein standard onto a 12.5% SDS gel, and run with a constant voltage of 80 volts.
After the gel run is complete, transfer the proteins from the SDS gel to a nitrocellulose membrane. After the transfer is complete, place the blotted membrane in a box, and incubate it for an hour in blocking solution, under gentle agitation. Then, wash the membrane three times for 5 minutes with PBST.
To detect the proteins, add the first primary antibody at the indicated dilution to the membrane, and incubate it overnight at 4 degrees Celsius with gentle agitation. The next day, wash the membrane with three 5-minute PBST washes, then, incubate the membrane with 20 milliliters of secondary antibody with gentle shaking for 1 hour at room temperature.
Wash the membrane three times with PBST again. After discarding the last PBST wash, add approximately 1 milliliter of horseradish peroxide substrate to the membrane, and detect the chemoluminescent signal.
