Does anyone have any experience in extracting hormones from saliva? I am at the stage of wondering whether I need to use protease inhibitor with or without EDTA.
As we know EDTA is a chelating agent which will bind to metal ions, but some of these metal ions will also inhibit protease activity which is what I want. But I have read others' post saying is good to use EDTA-free protease inhibitor. Is it because it will interfere downstream process? Any other reasons?
Saliva is known to be full of proteases and peptidases(and hydrolase in general), I would Highly recommend to use protease inhibitor cocktail sold by Roche or sigma. EDTA is not required if you are already using the protease inhibitors (as I assume you would use it to achieve the same goal)... Then In order to concentrate the enzymes contained in saliva, I would recommend a protein precipitation protocol such as TFA precipitation. Alternately, you can use a chloroform:methanol extraction such as the MPLeX method for the protein extraction and concentration http://msystems.asm.org/content/1/3/e00043-16. For the following steps it really depends on what type of experiment you are planning to perform gel-based-digestion, in liquid digestion, FASP ...
High concentrations of EDTA will affect purification using IMAC.
THanks Geremy for the article and comment, if there is a choice I prefer to isolate metabolites and nucleic acids together :)
Thanks Omar, is it because of the metals (nickel) used ?
I'd be curious to know a method for metabolite and nuceic acid all together...
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