I ran an ELISA (using the R&D systems duoset kit) last week for IL-1B on tissue lysate from the mouse dentate gyrus. I did not know how much IL-1B I was expecting to measure so I followed the kit instructions completely which recommended a high working conc of my standard at 1000pg/ml from which I then prepared my serial dilutions down to 15.625 pg/mL. I assayed 100ug protein in a 100 ul volume per well. My standard curve worked out perfectly and I got a good r2 value. My samples however appear to have very little IL-1B present and from looking at the absorbance values of the standard curve and the samples I reckon the the IL-1B concentration is <10 pg/mL. Looking at another study which is similar to my own, they also got very low IL-1B levels.
To repeat the ELISA Ihave two questions:
1) Is 100ug protein too high to use in the assay if I am attempting to detect such a low concentration of protein? Should I try 10 or even 1 ug protein per well?
2) I reckon my standard curve is too high given the level of IL-1B apparently present, how about preparing a serial dilution from 100 pg/mL (or even less?)