We have a commercial kit from RayBioTech to determine IgE from mice serum. In accordance with the supplier's instructions, we have to use a dilution of 1:10000 of the serum.
Is this dilution the most adequate? Should we try another one?
We only have one plate, and we cannot do experiments to determine the exact dilution to use. We want to nail the dilution from the get-go, thus we are seeking your advice.
Thanks, in advance, for all your help.
You should not change the original protocol since the dilution rate is too high that means the assay is quite sensitive. This is my first time I have seen such high dilution rate, I used to work with 1/101 or 1/1000 ratios for human samples. The highest concentration of the standard is 400 ng/ml, if your sample is out of the measurable range, you have to dilute more but the insert package doesnt say anything about that situation.
Can you use a few wells on the plate to test your intended dilution in order to see if it will work? Otherwise you're risking your samples and your plate.
With such high dilution factors, some of your sample proteins also easily might adsorb to the walls of the vials used for the dilution. Is there anything included in your buffers that will prevent/minimize adsorption, like BSA or a nonionic detergent?
Thank you Wolfgang Schechinger for your suggestions! We are thinking of doing a test column with several dilutions, alongside a calibration curve, to see what is best. Due to the fact that we only have 1 plate, we managed to think of the experiment like this, to not compromise our other samples. From what I read, I don't know if there is anything of the sort (BSA or nonionic detergent).