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.
