Electrotransfer proteins for Edman sequencing

Emir REMOVEem at unforgettable.com
Tue Nov 6 09:59:28 EST 2001


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
successful solutions.

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.

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.

-Emir


"D.K." <dk at no.email.thankstospam.net> wrote in message
news:9rvt6u$ih4$1 at news.doit.wisc.edu...
"Emir Khatipov" <khatipovNO at NOuchicago.edu> wrote:

..
>Hello All,
>Could someone recommend me a good protocol for SDS-PAGE and subsequent
>transfer of proteins onto PVDF membrane for subsequent Edman sequencing?

Only one important rule: Have a lot of protein in the band :-)
If you don't have that and if a band is clean, save yourself time and
send it to mass-spec-based sequencing.

>Which PVDF membrane did you use

Does not matter. They all work and if some does not work, the main problem
is elsewhere.

>how did you deal with non-polymerized
>acrylamide that can modify proteins and impair sequencing,

Almost never a problem. Don't deal with it before you know you have
a problem. Short pre-run of about 5-10 min is usually all it takes.

>what transfer
>buffers worked for you best, etc. Should I use PAGE buffers different from
>Tris-glycine (Borate?), and do I have to care about glycine on Page stage
at
>all?

No you don't. If you have enough common sense to wash your blot in water,
you are not going to have a problem.

>I know that CAPS buffer is a good alternative for Tris glycine transfer
>buffer, but I am concerned that high pH of the buffer (pH 11) will cause
>modification of side chains of amino acids.

Well, if you are that paranoid, you can always use CAPS at pH ~ 10
or you can use borate at ~ 8.5 or carbonate at ~ 9.0.

>In other words, I good detailed
>working protocol would be very much appreciated, or pointers to published
>resources.

Umm, did you also fall for that one-size-fits-it-all cookbook philosophy?
:-)

Regards,

DK





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