[Protein-analysis] Re: Glycerol during protein purification and protein oligomerization state

Dr Engelbert Buxbaum via proteins%40net.bio.net (by engelbert_buxbaum from hotmail.com)
Wed Jul 25 15:16:42 EST 2007

Am 19.07.2007, 22:13 Uhr, schrieb Emily Arturo <ecgarturo from gmail.com>:

> 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%!

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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