"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
