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Blocking for 1h vs overnight
What is the difference between blocking western blot 1hr at room temperature vs overnight at 4oC? Which gives better blocking?
Also, do you use Tween in the buffer when you block?
Re: Blocking for 1h vs overnight
Tween is generally a good idea to help prevent non-specific adsoption of protein to the membrane and wash away loosely bound proteins that can cause non-specific binding. Typically Tween is just added to the wash buffers, but at low concentrations it can help when added to the primary or secondary antibody as well.
Blocking time and temp depends on your antibodies and the type of blocker being used.
If you get really dirty blots when blocking at RT for 1 hour, try overnight at 4C.
If you still get dirty blots, you probably need to change your blocking protein or optimize your antibody concentrations. It might also indicate that it's time to change the pads you use during the transfer.
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