ELISA modification - (Apr/08/2012 )

Hi there,

I've done a number of ELISAs previously with the below protocol:

1:Coat plate with 1ug/ml protein
2:Leave at 4C o/n
3:wash
4:block/incubate
5:wash
6:add antibody/incuabte for 3hrs
7:wash
8:add secondary antibody/incubate for one hour
9:wash
10:add TMB
11:add HCL
12:read plate

So I am thinking at step 1: add my "receptor of interest" and then at step 6: add my protein that I wish to see if it binds to and then continue on with same steps...has anyone done a similar protocol before?...Ideally how much second protein do I need to add, would I just need to titrate out different amounts?

duhh,,,i just reread my post and realised the above didn't make sense AT ALL!...

so basically I have my protein of interest and I wish to see if it binds to a certain receptor...I need to do an ELISA to see if it does and I have done a number of ELISAS in the past using the above protocol...this one is different though because as oppsed to protein, antibody, secondary antibody..I will have protein, protein, antibody, secondary antibody..so my question is what steps would I have to modify I'm thinking
at step 1: add my "receptor protein of interest" and then at step 6: add my protein that I wish to see if it binds to and then continue on with same steps...has anyone done a similar protocol before?...Ideally how much second protein do I need to add, would I just need to titrate out different amounts?

Based on what you said in your second post, this should work. And yes, ideally you would titrate the second protein to see if it binds in a dose dependent manner. Otherwise you could just have non-specific or background signals.