Hi,
I am trying to measure the content of methylglyoxal-dependent damage on arginine residues in my protein samples. I know an extensive protein hydrolysis must be carried out in order to detect the MG-H1 adduct, however the standard procedure (24-h incubation with 6N HCl at 110 °C) reduces the yield of the adduct recovery. Alternatively, longer incubations can be carried out for several days in presence of several proteases (pepsin, thymol, pronase E, aminopeptidase, prolidase). Can anyone suggest me which products to use and how to proceed exactly, please? Thank You very much in advance.
Stefano Falone, Ph.D.
Dept. of Life, Health and Environmental Sciences
University of L'Aquila - Italy
Hy Stefano
basically you can increase your recovery by reaching higher temperatures over shorter times (possibly under vacuum and with some antioxidants added).
Microwave treatment will further increase the efficiency of the reaction, and you might use simple capillary tubes sealed with a Bunsen burner to make your own hydrolysis tubes that would not require a vaccum pump.
I personally compared hydrolysis, under vacuum, with 6N HCl and 4N Methanesulfonic acid, for 4 hours at 145 °C and I had a good recovery of the tyrosines I was looking for.
Hope this helps.
Dear Leonardo,
thanks so much for Your kind reply. I'll try. I'll contact You if further help is required.
Happy Easter!
Stefano Falone
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