Adjust transfer times based on manufacturer’s recommendations and place an additional membrane in transfer to collect any protein that has potentially blown through the primary membrane. Larger molecular weight (~150-250 kDa) proteins will take a longer time to transfer than smaller molecular weight (~25-100 kDa) proteins.Set the voltage, amperage, and time based on manufacturer’s recommendations.Carefully remove any air bubbles present between the gel and the membrane with a roller, as bubbles will prevent proteins from transferring from the gel to the membrane.If gel front begins to exhibit a “smile”, reduce voltage.A 7.5% gel will run the fastest, a 15% the slowest. The run time for the gel will vary depending on the percentage of the gel. Allow gel to run until the dye front has passed through the gel.Run gels per electrophoresis apparatus’ manufacturer’s recommendations.Molecular weight ladder also must be brought up to same volume with the ‘blank buffer’ in multi-lane gels. In any empty lanes, load a ‘blank buffer’ consisting of lysis buffer containing 4X sample buffer at the same volume as samples. When using multi-lane gels, load equal volumes of sample to each lane to prevent lateral band spreading.For trough gel load ~0.5-1.0 mg of protein. For a multi-lane gel load ~5-50 µg of protein per lane. Using gel loading pipette tips, load cooled samples and protein marker onto gel.Assemble gels in the electrophoresis apparatus and add 1X running buffer to recommended fill level.When testing multiple antibodies using the same lysate, try using a stacking layer with one large trough instead of multiple lanes to maximize the number of strips available for testing.Add TEMED and 10% APS as last ingredients of gel recipe since they are responsible for starting the polymerization process. (Gel recipes and recommendations are listed below).
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