total protein extraction for FPLC

Cornelius Krasel krasel at wpxx02.toxi.uni-wuerzburg.de
Tue Jan 27 05:14:10 EST 1998


Heinz J. Schaefers <schaefers at sun.XuchcX.edu> wrote:
> I normaly use RIPA buffer (0.05 M Tris, 0.15 M NaCl, 1 %
> Triton x-100, 0.1 % SDS, 1 mM PMSF) to extract total protein
> from cells.
> The protein extracted this way works fine in Western or
> North-Western experiments.
> Unfortunately, even after desalting (using HiTrap desalting
> column, Pharmacia) and adjusting pH, I can't get any binding
> to HiTrap Q (anion exchanger) or HiTrap SP (kation
> exchanger) FPLC columns (Pharmacia).

This is odd. Some of the protein should stick to the ion exchanger, even
at 0.15 M NaCl.

How do you detect binding of the protein to your columns? (UV won't
work because of the Triton.)

--Cornelius.

-- 
/* Cornelius Krasel, U Wuerzburg, Dept. of Pharmacology, Versbacher Str. 9 */
/* D-97078 Wuerzburg, Germany   email: phak004 at rzbox.uni-wuerzburg.de  SP4 */
/* "Science is the game we play with God to find out what His rules are."  */



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