ELISA modification

[biology_06er]

Active Members

34 posts


Posted Yesterday, 10:12 PM

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 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?

Thanks,
b_06er

[Wallyanna]

Why do you have two proteins to add if you have one particular protein in mind for an antigen-antibody reaction? Are you trying to combine two proteins somehow?