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Glycerol during protein purification and protein oligomerization state

Glycerol during protein purification and protein oligomerization state - Protein Forum

Glycerol during protein purification and protein oligomerization state - Protein Forum


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  #1  
Old 07-20-2007, 02:13 AM
Emily Arturo
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Default Glycerol during protein purification and protein oligomerization state



I am experiencing difficulty with reproducing the oligomerization
state of a protein overexpressed and purified from E. coli, from prep
to prep. I have considered increasing the glycerol concentration in
the buffers to promote the dimer state over the monomer state (no
other oligomerization states are detected). The DNA binding protein
is likely not bound with DNA during these later stages of purification
as we have incubated the cell extracts with a cocktail of nucleases.

Does anyone have any suggestions? The buffers already contain 10%
glycerol; is there any reason (that I've not yet thought of) that I
might not want to increase the glycerol concentration further? I'm
ashamed to admit it, but I'm currently uncomfortable with more than
10% simply because I've not seen literature that cited a buffer
containing more than 10%!

Any other suggestions are welcome...

Thank you,
Emily.

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  #2  
Old 07-20-2007, 06:11 PM
Joe Doe
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Default Glycerol during protein purification and protein oligomerization state

In article <[Only registered users see links. ].net >,
"Emily Arturo" <[Only registered users see links. ]> wrote:



Is the concentration of the protein the same from prep to prep? If the
concentration varies (say better or worse induction) perhaps that alone
is sufficient to favor monomer vs dimer from prep to prep.

You could also try purification strategies that favor keeping high
concentrations if you want dimer or the opposite if you want monomer.For
example gel filtration tends to dilute, ion exchange concentrate etc.

Other possibilities could be subtle changes in buffer composition or
conditions that favor or disfavor oligomerization - changes in ionic
strength, pH, redox state etc.

Roland
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  #3  
Old 07-25-2007, 08:16 PM
Dr Engelbert Buxbaum
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Default Glycerol during protein purification and protein oligomerization state

Am 19.07.2007, 22:13 Uhr, schrieb Emily Arturo <[Only registered users see links. ]>:


It should be quite possible to increase the glycerol concentration
further, many proteins are stored in 50% glycerol at -20 degrees.
Remember, Glycerol reduces the available water concentration and therefore
protein conformational freedom. This increases stability, but (reversibly)
reduces enzymatic activity.

I would however suspect that removal of the DNA could be the culprit: many
proteins become unstable if their ligand is removed. Perhaps adding short
DNA fragments, which are less likely to interfere with purification than
an entire bacterial chromosome, would help.
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  #4  
Old 07-26-2007, 05:51 AM
Frank Küster
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Default Glycerol during protein purification and protein oligomerization state

"Dr Engelbert Buxbaum" <[Only registered users see links. ]> wrote:


Or even just high concentrations (100 or 200 mM) of phosphate buffer. I
know about an (unpublished) DNA-binding protein which can be triggered
to fold in a stopped-flow by mixing protein solution with phosphate
buffer.

Regards, Frank

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glycerol , oligomerization , protein , purification , state


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