Coating Protein A and G on ELISA plate - (Sep/12/2008 )

Hi all,
I am currently trying to make the signal strong from optical ELISA by coating the maxisorp
plate with protein A first, then load my primary capture antibody on it.

I know that protein A works best at pH 8 and protein G work best at pH 5. I wonder will
I mess up the experiment, if I use standard carbonate buffer at pH 9.6 to coat the protein A/B
at 4C ovenight.

Thanks!!

No, I don't think so...

"protein A works best at pH8" means it binds strongly to Ab at pH8.

The carbonate buffer pH9.6 is used to increase the binding of protein A to the well.

If you are worry about messing up the experiment, you may use PBS for coating buffer.


Hope this may help.

i agree with minnie,

if the pH 9.6 is higher than the pI of protein A then u will have efficient binding. if the pH of the buffer used in coating is less than the pI of protein A, then you will reduce the efficiency. but at the end the relative difference in result does not change in an experiment.