how to denature protein without SDS and precipitating - PNGase A digestion help!!! urgent! (Oct/12/2005 )
I have to denature my protein extract before I do PNGase A digestion. this enzyme is inactive in SDS,
I tried to use NaSCN or high concentration (2%) of Triton X-100, both resulted in precipitation of lots of proteins.
What should I do to keep proteins soluble yet not precipitating without SDS??
Thank you.
Ann
-annh-
QUOTE (annh @ Oct 12 2005, 09:18 PM)
I have to denature my protein extract before I do PNGase A digestion. this enzyme is inactive in SDS,
I tried to use NaSCN or high concentration (2%) of Triton X-100, both resulted in precipitation of lots of proteins.
What should I do to keep proteins soluble yet not precipitating without SDS??
Thank you.
Ann
I tried to use NaSCN or high concentration (2%) of Triton X-100, both resulted in precipitation of lots of proteins.
What should I do to keep proteins soluble yet not precipitating without SDS??
Thank you.
Ann
I have never used this method, but you may want to check the following link using Urea as a denaturing agent http://www.pnas.org/cgi/content/full/101/17/6433 the paper deals mainly with TMAO, but it can give you an idea of the Urea treatment
Maybe others on this board have experience using urea...
-Rafflez-