Time for coating - (Jan/03/2011 )
I would like to know about time for coating wells: some people coat overnight others coat untill 48 hours.
So what´s the major importante in time of coating wells? For high binding plates, why this time variated so much? Protein weight?
Thank you very much.
Some people just have too much time for coating ELISA plates . Perhaps they always started on Friday and performed the ELISA on Monday. OK seriously, Corning has a really nice manual about coating the plates (am attaching to this post). You can find most of the relevant info there. No need to go overboard with the coating time.
It's one of those dogmas where the standard protocol says to coat/incubate for x number of hours and optimizing for each assay takes too long, so we figure that overkill is better. However, sometimes overkill is worse for your assay. If the antibodies have a high dissociation rate, then the longer the incubation, the less sensitive your assay will be.
i think it depends a lot on the type of plate u are using and your coating antigen... i usually coat proteins for checking anti protein antibodies in serum samples and i have observed that some of my proteins get coated within a variation of 5% within wells in less than a couple of hours and others take overnight time.... i think generally people use the ovekill stratergy as it was quoted earlier but personally i think it should be studied more cause sometimes coating becomes the most important step in your ELISA!!!