ELISA results - after adding substrate all wells are dark yellow (Nov/02/2009 )

Hi..
please tell me why my ELISA plate directly gives yellow color after adding the substrate

Which substrate and which secondary antibody do yu use ? Peroxidase labeled or Phosphatase ?

You may not have washed the enzyme off the plate.

Could be a problem of substract.
When you prepare substracte, can you see background on substracte solution before add it in the wells??

pesji on Nov 2 2009, 12:56 PM said:

how are you washing your plates before adding the substrate? most detergents interfere with chromogenic substrates so you need to wash your plates with dH2O a couple of times at the end before developing your plates

Thanks for your all replys,yes my problem is in my substrate and washing procedure