gray extract color
rapr at MED.PITT.EDU
Wed Sep 7 11:45:31 EST 1994
> To: yeast at net.bio.net
> From: Arnoud Kal <KAL at amc.uva.nl>
> Subject: gray colour extract
> Date: 6 Sep 1994 17:24:55 +0100
> I am trying to break large amounts of yeast cells with glass beads
> in a Sorvall Omni-mixer or in a Waring blender. The problem is
> that the cell go in as a brownish suspension, but after mixing and
> blending the cell extract has a gray colour. I never see this when
> I make extracts in eppendorf tubes. Can it be the buffer in combination
> with the stainless-steel container or knives? The buffer contains:
> 200mM Tris pH8
> 400mM Ammoniunsulfate
> 10% glycerol
> protease inhibitors
> The ammoniumsulfate is included because I want to purify a DNA binding
> protein, the salt elutes it from the DNA. Can it be left out of the
> breaking buffer and added after breaking the cells?
> Other suggestions how to break 250-500 grams of cells are very much
> appreciated. I used a Dynomill before (it worked) but this was not
> in my own lab and at that time I had to break 2kg cells.
> Arnoud Kal, University of Amsterdam, The Netherlands.
I had posted an unnecessarily brusque and arrogant reply to this request for
help, for which I apologize here. In fact Arnoud Kal does well to notice
changes in colors, etc., during his work, since these can give essential
clues to potential problems. The point of my prior post, that the color
change is notable only if there were a poor yield of the desired protein,
is a half-truth, since the quality of the desired protein may in fact vary,
depending on details of the extraction that could affect color also.
In Arnoud's case, it is possible that metallic contaminants from the stainless
steel (Cr, Mn, Cu, Fe) could account for the coloration; certainly I would
expect that blending with glass beads would abrade the steel, and the NH4
of the buffer is a powerful complex-ion former that could solubilize metals.
If so, then the heavy metals could interfere with enzyme activities and
generally mess up the extract (although they might also improve the yield
of some proteins, who knows). I suspect that omission of the NH4 might
help, so far as metallic solubilization goes, unless the beads have already
done the damage by abrasion, so late addition of NH4 simply solubilizes
after abrasion rather than during abrasion. I wonder if the color has
nothing to do with metals, but just reflects a much more concentrated
extract (due to better breakage) compared to previous types of extractions.
I would want to have some quantitative estimate of the % breakage, and
more importantly, the yield (and quality?) of the desired protein, before
worrying about tracking down the cause of the color change.
rapr at med.pitt.edu
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