At 09:48 AM 8/18/2000 -0600, Ting Wong wrote:
>I have encountered incomplete digestion of protein using trypsin. Since
>I was given the impression that trpytic digestion of protein can be done
>in mild denaturing condition such as 1-2 M 0f guanidine hydrochloride, I
>tried that but was not successful. The trypsin seemed to lose activity
>even at S/E (w/w) of 5 to 1 (20:1 was used in normal codition). I wonder
>if there is anything that I am not aware of in applying such condition
>for trypsin. Thanks in advance for your advice.
Ting,
Another myth -- often perpetuated in lists of proteases -- is that trypsin
is stable to such concentrations of trypsin. We did a few experiments to
assess that point because we had virtually the same experience as
you. Those experiments simply confirmed our initial impression -- trypsin
doesn't die completely in 1 M guanidine, but it's seriously injured. The
rate of peptide bond hydrolysis drops substantially, some bonds which
should be cut not act resistant, and cleavage rarely goes to completion.
Rod Levine
NIH
Bldg 3, Room 106 MSC 0320
Bethesda, MD 20892-0320
email: rlevine@nih.gov
voice: 1 (301) 496-2310
fax: 1 (301) 496-0599
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