I need to precipitate protein extracts in order to perform a derivatization reaction of carbonyl groups with 2,4-DNPH. According to the old original protocol everyone is referring to, protein extracts are precipitated with TCA and resuspended in 2N HCl /10mM 2,4-DNPH.
My troubles: After TCA precipitation I can by no means resuspend proteins in HCl/DNPH, absolute plastic! I tried with a different precipitation (Ethanol/Methanol/Acetone 50-25-25) but I had more plastic..
Question 1: Maybe the problem is not the precipitation itself but resuspending in HCl?
Question 2: does anyone know what is 2N HCl for in the derivatization reaction (Brady's test)? Do I need it?
Question 3: The same reaction in small scale is easily performed with the oxyblot kit in fc 3% SDS. Should I try to resuspend pellets in 3% SDS adding the HCl/DNPH mix in a second time?
Thanks for help and suggestions
unsoluble precipitated proteins