mysterious lysozyme or what?

kaj.stenberg at helsinki.fi.invalid kaj.stenberg at helsinki.fi.invalid
Sat Jan 17 07:52:45 EST 2004


Using lysozyme for reference I came across something odd.

My construct of interest has a calculated Pi of 9.2 (not experimentaly
checked). To test a modified protocol I used lysozyme as a model (Pi
9.7). The results puzzles me. 

In both cases buffer 20mM Tris, 2mM ZnCl2. A is 50 mM NaCl, B is 1M
NaCl. Ph 7.5 adjusted after everything added.

Material:
Sigma Lysozyme. Vial at work, from memory: "purified via 3 x
crystallization, 95% protein".

The puzzle:
1) Q-sepharose (anion) 
- Nothing through vith A-buffer. Gradient with B gives two distinctive
peaks (1:1 is 80% in height). Odd, but not unheard of. 

2) SP-sepharose (cation)
- peak immediately, but also peak when B-gradient starts. Both peaks
corresponds in height to one of the the Q-peaks. ???

(I should add that the identical program was run to both columns before 
adding saample in order to clean the columns, flatline then).

Unfortunately I did not collect fractions. But the original material
showes (highly overloaded, coomassie) another band, estimate 1/20 of the
major band (clearly larger, slower. Again, data at work).

As for now I am only interested in the quality of my columns, and the peaks
loocked individually nice. But I do not understand the results. Can anyone
help me?
(If nothing emerges I _will_ contact Sigma on monday regarding the
lysozyme prep).


-- 
Kaj Stenberg PhD
Department of Bioscienses
Division of Biochemistry
P.O. Box 56
00014 University of Helsinki
Finland



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