[Protein-analysis] Re: Glycerol during protein purification and
protein oligomerization state
Dr Engelbert Buxbaum
(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.
More information about the Proteins