mysterious lysozyme or what?
PC at no.email.sorry
Sat Jan 17 12:04:13 EST 2004
I find it very strange that lysozyme would bind at all to Q sepharose
(not that I tried it myself). Can it be that the real peak actually did
not elute from SP yet?
Are your columns fresh and clean? If not, wash them in 8M GuHCl.
Or NaOH as APB recommends.
The higher band is most likely egg albumin, which should have low pI and
will bind to anion-exchange.
And finally, are you sure that the vial that you used for chromatography
was actually chicken egg lysozyme and not chicken egg albumin? (please
do not take offence, it is just another thing to check). Sigma sells a
spectrum of albumins, crystallysed.
kaj.stenberg at helsinki.fi.invalid wrote:
> 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.
> 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).
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