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in this video we will discuss considerations for optimizing incubation time and dilution of primary antibodies when performing a western blot commercially purchased antibodies should have a starting dilution recommended by the manufacturer plan your dilution series around the manufacturer recommended dilution for example if a one to one thousand dilution was recommended make and test a two-fold dilution series ranging from a one to two hundred and fifty dilution to a one to four thousand dilution this should help establish the optimal dilution for your specific target protein the optimal primary antibody dilution has been established you can optimize incubation time the typical recommendation for primary antibody incubation is one to two hours at room temperature or overnight at 4 degrees celsius the optimal choice depends on your antibody and sample for example if the target protein is abundant in your sample a 1 to 2 hour incubation at room temperature
