Hello Everyone,
I usually block my membrane in BSA blocking buffer for 1 hour before incubating in primary overnight. This time i was pressed for time and decided to block at 4C over night. Please can anyone advice me on how long to incubate the membrane in primary antibody at RT.
Thanks
You have alternative for every step which does not interfare with other step. You can proceed for next step as usual. Either overnight at 4 degree, or 1-2 he at RT.
Hi, blocking overnight at 4C basically has the same effect as 1-2 hours at RT, so it should make no difference. The incubation time of the first AB is dependent on how good the AB is and which protein it detects. If you usually incubate at 4C overnight, it would be the safest to do that here also. Good ABs of very abundant proteins like Actin can also be incubated for 1-2 hours at RT.
You have alternative for every step which does not interfare with other step. You can proceed for next step as usual. Either overnight at 4 degree, or 1-2 he at RT.
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