>From the respondents already, it's nice to see the cleaning trick catch on.
To give details: wash PVDF sample in 1ml of MeOH containing 1ul of
triethylamine (Pierce), which pulls out the coomassie. The tube is inverted
maybe 10 times over 15 secs. The MeOH is aspirated and followed by 2x 1ml
MeOH in the same way. This procedure will remove SDS so we only get blocked
transfer lines only 1-2 times a year; some 70 percent of our 550 samples
per annum are blotted. No evidence of significant protein loss.
I can't imagine that the Coomassie has anything to do with your protein
degradation, but you could now test that to be sure. I hardly think that
there is anything such as an 'average' protein, and the background you get
will be sample dependent. Particularly bad samples might benefit from
dropping the cartridge temperature a few degrees during the cleavage step
and live with increased lag.
Does PVDF create a background? Sure, but it shouldn't be too significant. I
can only recommend that you try a 'blank' piece of whatever batch you have
and compare that to a 'blank' area on a blot. We use ProBlott most of the
time, but Immobilon PSQ seems similar in terms of cleanliness.
Expect to see little or no response at glycosyl residues. Some people
comment on the production of dehydroalanine at Glyco-Ser, but I'm not
convinced it is always observed.
Len
*********************************************************************
Dr Len C. Packman
Assistant Director of Research
Protein and Nucleic Acid Chemistry Facility
Department of Biochemistry
University of Cambridge
80 Tennis Court Road
Old Addenbrookes Site
Cambridge, CB2 1GA, UK
Tel: +44 (1223) 333639 (including answerphone)
FAX: +44 (1223) 766002
e-mail: lcp2@mole.bio.cam.ac.uk
Visit my WWW page at http://www.bio.cam.ac.uk/proj/adr/PNAC/pnac.html