I began sequencing in order, eg band 1 , 2, 3 ...
Band 1 gave usable sequence, but it had a very large amount of background
of all amino acids, not all equal, but within about 30 % of each other.
The background of the average amino acid was easily 8 pmol and did not
wash out with increasing cycle number. This suggested to me that the
sample was cleaved internally and I was getting noise from 'background
sequencing'. Band 2 being fainter, I expected lower background, but I
still got a substantial amount, and the signal for any "real" sequencing
signal was so low as to be impossible to read.
After the experience of bands 1 and 2, I decided to wash band 3 before
sequencing it. I wet the dried PVDF with 500 microliter of 50%
methanol/water, and rinsed several times with water.
There was no substantial background and I was able to get sequence info
sufficient to id the band as BSA. Obviously, I have no proof that
background would have been present in this BSA band if I hadn't washed it,
but it didn't seem to have hurt anything either. If the background in
bands 1 and 2 was due to internal cleavage sometime during purification,
why wouldn't the cleaved products resolved/smeared on the gel... the bands
looked prety good. If the cleavage occurred during transfer, then why
didn't BSA get internal cleavage also?
Sorry if I've rambled a bit here.
Best regards,
Matt Williamson