We are trying to optimize ELISA protocol for the first time in lab. Experts please help me.
We purified antibodies that were raised using a peptide. When we do ELISA with peptide, it gives dose dependent signals, but with protein it does not give any signal .
Earlier we were using native protein, we thought may be the epitope is hidden, then we tried denaturing the protien using 2% SDS, however the attempt was unsuccessful. I also tried acetone precipitation after denaturing protein to remove SDS. But that also dint work.
Concentration of protein and peptide was same, tried with higher conc. of protein also, but all failed.
Please give tips/ideas, what should we do.
We know the protein works, as we have seen the band in western blot.