Establish of an indirect ELISA in one month possible - (May/03/2013 )
during my thesis i want to quantify the if Histon 3 or 4 gets more methylated\acetylated in a specific tissue of mice living in different conditions. Therefore we have several Antibodys for both, ELISA and Western-Blot. First we tried Westernblot and it works fine but we need to much of the sample (10 ug per blott) and the material is very limited (down to 60 ug per sample). Furthermore stripping didnt work because we lose to much of the proteins or the signal is still there. Therefore we think about to establish an indirect elisa in our lab but the problem is i have only two months to go for my diploma thesis. No one done ELISA before in our lab so iam on my own.
I want to ask if is hard to establish an indirect ELISA (the antibodys are working very well for western blot) and how much protein from a whole tissue is per detection necessary ? Abcam recommend 1ug per well, is this right ? Is ELISA suitable to detect and quantify very fine methylation\acetylation changes of Histons between different samples ?
I hope somebody can answer me this basic questions. Thanks !!!
we use up to 30 ug per well in western blot. western could be as sensitive as ELISA. You can't really say which one is better. Getting a good signal depends on many factors. It depends on the antibody too. I understand your problem is with the quantity of sample, but I would still go for western, because I always have problem with last step of ELISA when I add substrate.
I run ELISA's all the time. An ELISA protocol can be set up in that amount of time if you have the proper controls but if you want to quantify and you have limited samples, you should consider sandwich ELISA's. Do you have two compatible antibodies to use for a sandwich ELISA? If so, you can easily (and for not much $$$) biotinylate your tracer antibody and increase your sensitivity.