Please help - FACS to ELISA for antibody testing - (Mar/01/2012 )

Hello all, I am coming back to this forum after a big gap. I normally assay my cels and clones for expression using FACS but as we are studying novel proteins, the commercially available antibodies are all not for FACS application. Many are for ELISA application. Can some one give me a protocol how to assay the cells for protein expression using elisa? assuming that the protein we are studying is expressed on or inside the cells and NOT a secreted protein.
I did ELISA many times before but for secreted proteins - I mean cell based (like the hybridomas) and even estimated the expression titer. But now I have the cells which express proteins ON the surface and I need to assay with a commercially available antibody using ELISA.
To start with - how do I coat the plates?


Thanks for your replies.

ps: I also posted this same post in the ELISA group of this forum but no answer.

will this link help? http://www.celprogen.com/DS/K36094-21A%20-%20DS.pdf

Hi Thanks for replying. I went through this but it looks like it is a kit meaning everything is highly specific made for that assay ONLY right?

I am trying to find out how to assay my protein of interest using ELISA when the protein is ON the surface of the cell. Through online research, I understood that we need to lyse the cells. Looking for more info.
thanks.

You can follow their procedure using your cells and substitute your antibodies. I am unfamiliar with the matrix on the surface of the plates but you could talk to them to see if your cells would grow.

Alternatively, you could vigorously wash your cells, dialyze, concentrate the wash and test and/or lyse, dialyze, concentrate and test.

A question before starting is what concentration mg/ml, ug/ml, ng/ml, pg/ml are you expecting? Can you obtain some of the protein to test your elisa? You will need some sort of positive/negative control to determine if your test works.