Amanda,
A few comments on nitrocellulose or PVDF digestions.
1) What is your application? LC/MS, MALDI-TOF MS, or HPLC isolation for
internal protein sequence data? For MALDI-TOF or direct HPLC you do not
need to use PVP-40 but should use a non-ionic detergent, octyl
glucopyranoside (0.5-1%) if you require MALDI-TOF and reduced Triton X-100
(1%) if you are going directly to HPLC. The detergent blocks the membrane
(hence no PVP-40) and elutes the peptides off the membrane with about
80-90% yield. I am unfamiliar with the LC/MS ramifications of the
detergents but I would guess they are not compatible.
2) As far as destaining, I agree with Axel to be careful about using strong
solvents for removing coomassie. PVDF-bound proteins can be destained with
up to 100 methanol while nitrocellulose samples have to be treated more
carefully. His suggestion of using 20% methanol should work OK.
3) Ziptips do cleanup samples very well for MALDI-TOF but I have noticed we
always lose a certain amount regardless what we do. Less than 50 ng we get
no recovery by MALDI-TOF (whether solution, in-gel or membrane digestion)
if a ziptip is used. Does anyone else experience this? When we use
octylglucopyranoside and ammonium bicarbonate (instead of tris) we can get
MALDI-TOF data however this is for PVDF samples which can be destained
easier. Nitrocellulose is more of a concern.
4) Although our lab pioneered some of the membrane digestion work in the
past 10 years (use of non-ionic detergents) we rarely analyze samples this
way any more. In-gel digestions provide much more useful data with the
high quality, high accurate MALDI-TOF data we can get and the lower volumes
we can use for capillary HPLC. We only perform PVDF digestions if
investigators need N-terminal data and need to isoltae longer peptides for
Edman sequencing.
I hope this helps you out.
Joe
Joseph Fernandez
Associate Director
Protein/DNA Technology Center
The Rockefeller University
1230 York Ave.
NY,NY 10021
Phone: 212-327-8869
FAX: 212-327-8620
Email: fernaj@rockefeller.edu
website: pdtc.rockefeller.edu
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