> I disagree. If you use "just enough" column capacity, not
> only it is not wasteful, but it frequently gives better recoveries
> for problematic proteins. I was purifying a protein for the same
In general, yes. For the purposes of 'high throughput' - no, one simply
can't afford to play with this kind of stuff for too long if one has to
purify >5-8 proteins per week (on one AKTA that is) (and that's what we
strive to get eventually).
> further concentrating step. The problem with this approach
> is that it is painfully long and requires more careful optimization.
Yup. You said it :)
> I noticed that! One batch that was purified with AS pption had
> different pH optimum than another batch that used Q column. Any
> explanation for this effect? (Heterogenuity introduced
> due to partial denaturation of complex proteins during pption
> would be my first guess).
Could be several things, including trace sulfate trailing on with the
protein. More likely formation of aggregates and subtle changes in
protein conformation.
> Yes, I believe 20V will do it even at low concentration of
> chelator. I _was_ surprised to find that 3V of 20 mM do not
> strip (100 mM did right away).
Hm. What resin are you using ? We use Superflow Ni-NTA from Quiagen (I
call it the rip-off resin $700 for 100 ml, come on !)
> Thanks for the info about proline. I always put Gly, but perhaps
> Pro is a better idea.
Well remember that the more you put on, the more chance to influence
xtallization you get. We have done a little study on the effects of
protein tags on xtallization and there clearly is *an* effect, though it
is not clear-cut 'bad' that is in our experiment tags changed protein
crystallization conditions, space group etc. but the data were as good
or better in many cases. In some cases, tags killed xtallization
completely, though.
> :Proteins are very different and one never knows what to expect.
> :"Standard Protocols" barely ever work in protein purification on a large
> :scale :)
> Oh yes, sure. That's the beauty of working with them.
Yes, as long as one gets to do something else as well - all purification
can get very tedious, especially if things do not work out.
Artem
--
|Dr. Artem Evdokimov Protein Engineering |
| NCI-Frederick Tel. (301)846-5401 |
| FAX (301)846-7148 |
|eudokima at mail.ncifcrf.gov |
|http://www.ncifcrf.gov/plague |
