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Incubating the filters two overnights with primary antibody. - WESTERN BLOTTING (Sep/09/2005 )

Is that okey that I incubate the filter with primary antibody two overnights before adding secondary antibody for detection? This is because I do not have the equipment to detect the signals in the weekend so I have to detect it on a Monday. Due to it I incubate the filter with primary antibody on Saturday till Monday morning and then detect the filter on Monday. Would this give me problems?


Just do and see, what this would bring

As for me, I incubate 1hour with the 1st Ab, 30 min with the 2nd, so all the same day


I know that some people do it. So although I would not recommend it as a standard procedure, apparently it works.
However, I would never do this for phospho-specific antibodies, since phospho-groups might be removed easily by phosphatases during the incubation (despite the presence of phophatase inhibitors).


I've had to do this back when rotating through labs at the beginning of graduate school. It worked ok - just ensure that the incubation is rotating gently at 4 degrees.