Have I used too much protein in my ELISA? - (Feb/24/2014 )
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?)
1) You should run multiple different dilutions of your lysate, ex: 100, 50, 25, and 10ug total protein on the same plate. Use the dilution buffer provided in the kit. If you get the same IL1B concentration result after correcting for dilution factor on all 4, then you can be certain you have an actual measurement. Otherwise, repeat again with higher dilutions.
2) If the ELISA kit states that the assay range is 1000-15.6 pg/mL, then you should use that only. Lower standard concentrations may not give enough signal above the background.