What is the main use of EDTA in western blotting. Can someone please elaborate. I know it is a metal ion chelator. But any other specific reason for its use in western blotting?
"Alkaline phosphatase (AP) and horseradish peroxidase (HRP) are the two enzymes that are used extensively as labels for protein detection. Alkaline phosphatase, a 140,000 dalton protein that is generally isolated from calf intestine, catalyzes
the hydrolysis of phosphate groups from a substrate molecule, resulting in a colored or
fluorescent product or the release of light as a byproduct. AP has optimal enzymatic activity at a basic pH (pH 8-10) and can be inhibited by cyanides, arsenate, inorganic phosphate and divalent cation chelators, such as EDTA. Please see the attached literature downloaded from internet (page 11)". This may partly answer your question.
If you are talking about EDTA in your lysis buffer - it is to help inhibit metalloproteases which require divalent cations to work. When you lyse your cells you want to inactivate proteases present in your sample to prevent other proteins (e.g. proteins of interest) in your sample from degrading. This is the same reason you might add a protease inhibitor cocktail to your lysis buffer (which usually contain EDTA anyway).
http://www.sigmaaldrich.com/catalog/product/roche/CORO?lang=en®ion=GB
Sohail: Thanks, I used Ab conjugated with horse radish peroxidase.
Alex: Thanks a ton, that helps!
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