Protein solubility after amm.sulphate precipitation
wind at biobase.dk
Mon Apr 28 02:26:20 EST 1997
Chia Jin Ngee (mcblab47 at leonis.nus.sg) wrote:
: Koen A.L. De Smet (k.desmet at ic.ac.uk) wrote:
: : Malini Madiraju wrote:
: : >
: : > Dear netters,
: : >
: : > I have a unique problem. My protein doesn't to go into solution after
: : > ammonium sulphate precipiation even though it was soluble before
: : > precipitation.
: : >
: : > I do the following before amm.sulphate precipitation: Elute the protein
: : > from nickel affinity column, dialyse it into a buffer (8mM imidazole,
: : > 500mM Nacl, 10% glycerol, Tris-Hcl, 20mM, pH 7.5). Protein is soluble at
: : > this point. Then I do amm. sulphate precipitation. The ppt is now
: : > insoluble in the same buffer. I do ammonium sulpahte precipitation to
: : > concentrate the protein.
: : >
: : > I welcome any comments, suggestions or references that could help me
: : > solve this problem. Thank you.
: : >
: : > Malini R. Madiraju
: : I am not sure if this is related, but you may like to know this anyway.
: : I once had a protein expressed in the pQE system and it was soluble.
: : After purification, I froze the fractions in the buffer they were in.
: : (I cannot get hold of the details immediatly, because this was years
: : ago, but it probably was the buffer recommended by QIAGEN, for native
: : purification using imidazole elution).
: : When I took it out of the freezer and thawed it, most of it was
: : precipitated. I could get it soluble again by adding NaCl to 1M.
: : I assumed that it precipitated because it had not folded properly, but
: : was still soluble enough not to form inclusion bodies.
: Same problem here. Elution with imidazole resulted in precipitation of
: the protein. But elution with EDTA did not. Any explanation besides the
: protein not being folded properly?
: In my case, I didn't freeze the eluted protein solution. I refrigerated
: it at 4 deg C and it precipitated overnight.
: Jin Ngee, Chia
: (Genie, the OligoMan)
: mcblab47 at leonis.nus.sg
: Views and opinions expressed are solely mine and not of my employer's.
: Any further grievances can be settled over a Virtua Fighter 3 match.
Allow me to join the chorus, I have just purified a GST-fusion from a
GSH-column that appeared soluble when it came off the column, but ppt
after -80C storage.
Is there another way of dealing with this than the 1M NaCl solution? I've
tried 10%-25% glycerol without any luck. I really wouldn't like to have
to much salt in my preparation...
Second, Could someone explain what folding has got to do with it? I
realise that denatured proteins will have a greater tendency to form
complexes, but is it the only explanation for proteins with variations
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