I haven't done very many ELISAs and since we have no postdocs in our lab and my PI is strictly DNA/molecular-based, I'm pretty much trying to learn how to do them from mat/meths in papers (ha!).
My first problem is that I'm trying to detect (and quantify) one protein out of a mixture of proteins (plant soluble proteins). The protein is definitely there in high quantities (confirmed by many Westerns) but my ELISAs are not picking it up. I'm using alkaline phosphatase and I get some colour change after many hours' incubation but not much. My positive controls (purified protein of interest) always come up without any problems.
To further complicate things, I did some work on this protein a year or so ago and never had any problem picking it up. The difference now is that the tissue I'm working with has been freeze-dried and ground into a powder, as opposed to a TSP extraction from fresh tissue with carb/bicarb buffer.
Could it be that the freeze drying has altered my protein somehow and that it makes no difference for Western because my samples are denatured anyway before loading?
My second question is, sometimes my standard curve is a bit backwards, as in the lower concentrations actually give a higher OD than the higher concentrations. Could this mean I haven't properly optimised the Ab concentrations for my ELISA?
Thank you for any help you can give me - I'm stranded in an ELISA-knowledge-free world and I'll be grateful for any tips you can give me, however small.
Edited by vojera, 06 November 2009 - 07:38 AM.