Electrotransfer proteins for Edman sequencing
dk at no.email.thankstospam.net
Tue Nov 6 20:07:14 EST 2001
In article <AWSF7.78$N4.3944 at news.uchicago.edu>, "Emir" <REMOVEem at unforgettable.com> wrote:
>Look, DK, I appreciate your effort to reply, but I thought it was clear that
>I am not looking for the common sense recommendations (pre-run the gel,
>rinse the blot,... wear gloves) a more or less experienced biochemist would
>be aware of. I welcome feedback from people with first-hand experience and
I thought that's exactly what I provided (based on first hand experience).
Lets summarize: Have plenty of protein, use any PVDF brand, pre-run for
5-10 min only, transfer in anything you want (tris/glycine, CAPS, carbonate,
borate), but if you use tris/glycine wash membrane very, very thoroughly.
>Sequencing costs money ($200 for 5 cycles in our case) and it is not
>desirable for us to pay for attempts doomed to fail . Just-do-it philosophy
>(I understand this in your understanding is an alternative to a "cookbook
>philosophy" :-)) is not working all the time.
No, my understanding of an alternative to a cookbook philosophy is
understanding exactly what you are doing and why and adjusting things
you are doing based on this knowledge rather than on religious beliefs.
An example: that glycine thing. The same people who told you to not
run transfer in Towbin buffer do their gels by Laemmli. In other words,
they do transfer from gels containing glycine.
>The staff at the sequencing facility specifically pointed out that high
>glycine backgrounds due to using glycine transfer buffers and blocking of
>sequencing due to protein modification during PAGE are very common causes of
>failure. As for the quantity of the protein, I was told 50 pmol of my ~50kDa
>protein (i.e. ~50ng/band) would be enough.
Yes, 50 pmol is OK for Edman degradation, but - sorry - for a 50 kDa protein
50 pmol is 2.5 micrograms, which is exactly what I meant - A LOT. With 50 ng
you can still do it just fine by mass-spec.
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