Tris-tricine gel transfer help
Frank O. Fackelmayer
Frank at Fackelmayer.de
Mon Nov 19 12:54:15 EST 2001
"Kirk A. Gilbert" wrote:
> Here's my question: Is there an optimal transfer buffer to use with 15%
> Tris/Tricine gels and semi-dry transfer onto PVDF? Details follow:
> We're just beginning to use 15% tris/tricine gels (15x15cm) to anlayze a
> small (~10kd) protein from whole lung extracts (15 ug per lane).
> Previous expts. with 15% tris-Cl /glycine conditions gives us a broad
> band down in the area of our protein of interest.
> Our first attempt at Tris/tricine gels (whew! these gels take a long
> tome to run!)
in an minigel-format (8x5cm), we used to have running times of around
45min. I usually used 12%PAA, and it worked well down to 2-3kDa. No need
for 15%, and not at all for a protein of 10kDa!
> gave a very tight band, but after semi-dry transfer, a
> lot of protein was still in the gel. We used our standard
> Tris/glycine/20% methanol transfer buffer supplemented with 0.01% SDS.
> Transferring onto 0.2um PVDF. We've detected protein with subsequent
> Western blotting, but the entire molecular weight range of proteins
> remained in the tris-tricine gel.
> Any suggestions for Tris/tricine conditions, especially with respect to
> transferring would be greatly appreciated. Thank you in advance.
I used the CAPS transfer buffer described by Paul Matsudeira some years
back. Worked perfectly for peptides down to 3kDa. It is basically 10mM
CAPS, pH11.0, 10% MetOH. Remember to make up fresh each time, as CAPS is
instable at RT.
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