PVDF-Protein Sequencing
Gautam Sarath (gsarath@unlinfo.unl.edu)
Mon, 11 May 1998 17:16:32 -0500 (CDT)
Dear Deb: I have also noticed these large increases in background for some
samples, especially those that are glycosylated, and or come from odd
sources, eg rumen bacteria etc. I had always thought that the background
problem was confounded when a sort of dirty sample contained residues
recovered in lower yields, eg Trp, Cys, Pro, Ser etc. On a sample I was
analyzing yesterday, there was a dominant sequence in a sample with a high
background (sample signal approx 8 picomoles), there was no problems in
identiyfying the first 16 residues, 16th was a Pro and the sequence went to
zip after this cycle. I could not obtain readable sequence past this
point. I have noticed a similar large drop in plant glycoproteins blotted
and stained with C.Blue and with Amido Black. CBlue has more crud than
amido black. Although I do not know the answer to this problem, I too am
very interested in an explanation. One of the possible causes that Bob
Apflezweig at ABI mentioned was carryover from the flask in a random
manner, but this will never be consistent, and cannot explain loss of
signal. On glycoproteins, I would expect that there is some degree of
deglycosylation as a result of repeated acid/base exposure, whether this
could accelerate backbone breaks I do not know. Proteins should be
inherently unfolded on blotting, expect that many proteins (eg receptors)
maintain substantial structure on blots, since these beasties can be probed
with a ligand. In this scenario, would proteins that contain more
structure on PVDF degrade faster or slower than those that are essentially
linearized during SDS-PAGE followed by transfer? AHH the armchair
quaterbacking, apologize for no real explanations though, I know there are
some good ones out there.
I think you can remove excess CBlue from blots by quick soaking in 50 %
methanol with a drop of triethylamine.
Gautam
Gautam Sarath
N-226, Beadle center
Protein Core Facility
Department of Biochemistry
University of Nebraska-Lincoln
Lincoln, NE 68588-0664
Phone: 402-472-2928
FAX: 402-472-7842