High-Precentage Gel Grivance

I have been having a lot of problems running 15% acrylimide gels. I am finding that the two lowest molecular weight markers (15 and 10 kDa) are "seperating" into multiple (2-5) bands. The adjacent protein bands are also poorly resolved; the protein bands are not well defined bands but rather U-shaped with the protein appearing to pool along the edges of the lane. I have made new Tris buffers for both the stacking and seperating gel, tried using more and less TEMED but with no improvement. I make up fresh ammonium persulphate each time I run a gel. Oh yes, any other percentage gel I run (7.5, 10 and 12%) Any suggestions??? Thanks!

[Only registered users see links. ]

Try replacing glycerin with Ficoll and using buffer of 0.5 x or lower
concentration.
Emir

<[Only registered users see links. ]> wrote in message
news:[Only registered users see links. ].. .
finding that the two lowest molecular weight markers (15 and 10 kDa) are
"seperating" into multiple (2-5) bands. The adjacent protein bands are also
poorly resolved; the protein bands are not well defined bands but rather
U-shaped with the protein appearing to pool along the edges of the lane. I
have made new Tris buffers for both the stacking and seperating gel, tried
using more and less TEMED but with no improvement. I make up fresh ammonium
persulphate each time I run a gel. Oh yes, any other percentage gel I run
(7.5, 10 and 12%) Any suggestions??? Thanks!
[Only registered users see links. ]

....and please let us know if it worked :-)
Emir

"EK" <> wrote in message ...
also
I
ammonium

>I have been having a lot of problems running 15% acrylimide gels. I

Many years ago I ran fairly high percentage gels (13.5-15%).
Something I noticed for low molecular weight proteins was that I'd
often get multiple bands (when none were expected) if the sample
volume were too large (like more than 5 ul). You might try running
smaller sample volumes if you think this might be an issue.

Mike
--

Michael L. Sullivan, Ph.D
Research Plant Molecular
Geneticist
U.S. Dairy Forage Research Center
1925 Linden Drive West
Madison WI, 53706

(608) 264-5397 Phone
(608) 264-5147 Fax
---