I'm new to performing ELISAs on tissue lysate so please forgive this basic maths question!!
I've performed a Bradford on my samples and now I'm at the maths stage of figuring out how much protein to assay and how to calculate the necessary dilution.
For example, I diluted my samples 1:100 for the Bradford, from the standard curve one of my samples was calculated at 29.41 ug/ml which becomes 2941ug/ml when I factor in the dilution, and then converting this to ug/ul I get 29.41ug/ul. I obviously want to assay as much protein as possible ( I was thinking 100ug? per well) So for 100ug protein, I need to use 33.93ul of my sample, but for the ELISA, I need to add 100ul sample per well, so do I just make up the remainder (66ul) with RIPA buffer or will this dilute my carefully calculated protein concentration? Or have I gone about my calculations in a completely wrong manner?
Glad of any advice