The protocol for sandwich ELISA kit that I am using requires me to incubate the plate at 4 degree Celsius overnight after adding the biotinylated tracer. Provided there is no microbial growth and/or reduction in buffering capacity which some people think might be an issue with longer incubation times, does incubating plates over two nights at 4C increase/decrease the signal (or affect quantification) in case of colorimetric detection?
Thanks in advance!
Yes, it does! Signal is typically more probably because of nonspecific binding. Even too long of blocking is also not good and over blocking significantly affect the signal.
Hi，
I had incubated at 4C for one day and two nights, and the result was acceptable. So I think for 2 nights is ok for you.
But I can't guarantee it will be ok for more than 2 days. I think the signal will weaken, because of protein degradation. And another method - increasing the concentration of biotinylated tracer when plates incubated.
Good luck.
Hello,
I do agree with Mr. Snow. The intensity or signal will start to weaken with time. The best is to start after overnight incubation.
All the best.
I've coated plates on Fridays for use on Monday and its been fine - if its for a qualitative result.
I have also coated my ELISA for 1 hour at 37 degrees for same day use. This has worked fine also.
I wouldn't recommend but it depends how desperate you are to get things done quick
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