ELISA Recombinant Protein Coating

[gandalfthegrey]

Hello guys.

I'm new to ELISA and I would like to get some advice on the best method to coat different amount of recombinant protein onto ELISA plates.


I want to coat my plates with different amount of proteins starting with 0ng, 25ng, 50ng, 100ng and 200ng. However what is the best way to do this.

Should i do a serial dilution starting from 200ng, and dilute it or shall I prepare each amount of protein separately then coat it into the plate. Which method is better?


Thanks

[danfive]

I assume you're talking about recombinant proteins as standards. Not some kind of competitive or indirect ELISA. If so I've seen it done both ways, with a serial dilution before starting the assay performed by the user, or pre-diluted standards. The main concerns should be are proteins stable in the storage buffer? and will you lose protein due to sticky tubes? Especially for the low concentration standards, (most kits use glass vials).

[r15]

gan and danfive!
can u explain me in detail about the various steps involved in developing an elisa for detection of antibody fr an antigen... please! am new to this technique.. please help me..