At 16:04 02.08.00 +0000, you wrote:
>
> We intend to digest proteins in gel by using Lys-C and Arg-C
>enzymes. We then have the following questions:
>
> 1) Do these enzymes work well for protein identificatioin by peptide
>mass fingerprinting?
> 2) Are there any special protocols for Lys-C from Lysobacter
>enzymogenes (Sigma P3428) and Arg-C from mouse submaxillary gland (Sigma
>P 6056) or they work equally well when obtained from different sources
>and companies?
> 3) Could the gel be silver stained or should it be coomassie blue
>stained?
> 4) Could anyone send specific protocols for protein in gel digestion
>with the above Sigma enzymes?
>
> Thank you very much in advance.
I can't comment on the Arg-C, but we had good results with Lys-C from
Lysobacter enzymogenes sold by Roche Diagnostics (at least in Europe). You
can use almost the same protocol as for trypsin (we use a concentration of
12.5 ng/ul in 50 mM NH4HCO3 to reswell the gel and wash away all the excess
liquid).
In our experience almost all proteases will work within the gel if they are
able to enter the gel matrix. The smaller, the better. For this reason
Arg-C (55 kDa) will probably be a little bit troublesome, while trypsin (23
kDa), Lys-C (30 kDa) and Glu-C (27 kDa) work well within the gel.
The question regarding the staining is related not so much to the enzyme
but to the available amount of substrate. In my experience the high
sensitive silver staining methods using glutaraldehyde will give poor
results due to crosslinking of the proteins in the gel. The lower
sensitivity stainings without crosslinker (like the method from Shevchenko
et al.) work well with MS (even better after destaining according to
Gharadaghi et al.), but they show not very much higher sensitivity than
colloidal coomassie staining.
Yours sincerely,
Marcus
**************************************************************
Dr. Marcus Macht
University of Cologne
Centre for molecular medicine - Service laboratory
Joseph-Stelzmann-Str. 52
50931 Cologne, Germany
Tel.: +49 221 478-6995
Fax: +49 221 478-6977
e-mail: Marcus.Macht@uni-koeln.de
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