How to Make a Good Western Blot
The standard way to see the target protein is to let the primary antibody mix with the sample for one hour at room temperature. But an overnight incubation at 4°C should give the antibody-antigen reaction plenty of time, making it more likely that a positive signal will be shown. It also help to perform all washing steps at 4°C, that can be done using BlotCycle, automated western blot processor
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