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How do I prepare a sample to quantify a certain protein with ELISA? - (Jan/08/2007 )

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QUOTE (Ceri @ Jan 15 2007, 12:51 PM)
As WAstate says the sandwich ELISA uses an protein specific antibody coated onto the plate to capture the protein in the sample and a different species (or biotinylated) protein specific antibody plus a enzyme conjugated streptavidin or species specific antibody to detect it. In a direct ELISA the protein is coated onto the plate and the detected as above.

If you have a complex mixture of proteins the direct ELISA may be less specific. A sandwich ELISA should be more specific in this case.
You talked about using a total cell extract in which case you may be better off using a sandwich ELISA. So you need to have a second protein specific antibody from a different species or to biotinylate some of your first antibody (you can get commercial kits to do this). If this is a lot of hassle start with the direct ELISA coating the protein extract onto the plate and see how you get on. It might be worth thinking about negative controls to see how much background you are getting (e.g. a negative plant tissue, knockout seeds?, seed extracts deleted of your proteins using your antibody and protein G sepharose or seed extracts from a different but related species of plant).

All the best,

Excellent, thanks everyone for your help, i have quite a lot to research now! sounds like the sandwich ELISA is the best route if i have the materials, so i will investigate that further.
Thanks again everyone, i'm sure i will have more questions soon!


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