Re: Digest

Katheryn Resing (Katheryn.Resing@Colorado.EDU)
Thu, 29 Apr 1999 09:05:40 +0000

We get a lot of autodigestion of the V8 in in-gel digests with this enzyme,
so digest blank strips of gel to determine the amount of back-ground
peptides from the proteinase. We first determined the best V8
concentration using a standard (lysozyme run out on gels, at about the
total protein concentration of the sample we wanted to digest) with serial
dilutions of the V8 to determine the minimum amount of V8 required. We
washed the strips with buffer after swelling in the protease, in order to
minimize the excess protease. We reduced and alkylated the real sample
before running on the gel (rather than alkylating in the gel piece), but I
don't know how relevant that is.

Katheryn Resing