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Posted: Tue Feb 19, 2008 3:07 pm
by danko
Which in turn affects the protein conformation and solubility etc. etc.
Still the main message is as stated in my previous post: An easy start would be a low pH experiment/investigation.
Best Regards
Posted: Wed Feb 20, 2008 9:51 am
by HW Mueller
That´s exactly what one wants: Change the characteristics of the system to get the analysis to work. That´s what one does by going to low pH, as well.
Posted: Wed Feb 20, 2008 10:30 am
by danko
I’m not exactly sure what your message is, but low pH and high buffer concentration is not the same thing. They represent 2 completely different mechanisms/techniques. If you’re talking about HIC then that is another story. Also you might like to study Hofmeister a bit for additional info.
Otherwise I agree that both actions will introduce selectivity modifications and I believe I’ve stated it earlier (although indirectly) but the results will be quite different from one another.
Actually I’m willing to predict that high salt/buffer concentration in combination with a C18 column will get you into trouble (in protein context naturally). Even more so if you’re planning to run isocratic. So as mentioned earlier the things get a bit more experience demanding and that was my point.
If your mission is to say the last word, then please be my guest
Best Regards
Posted: Sun Feb 24, 2008 12:30 pm
by HW Mueller
My point was to innovate. (But: ask a protein, sometimes, whether it is happier with pH = 2 than with pH = 10, most likely it will depend on which protein you ask).
Posted: Sun Feb 24, 2008 3:08 pm
by Uwe Neue
But since most proteins are not happy with acetonitrile, why do we care which pH makes them happy?
Posted: Thu Feb 28, 2008 3:19 pm
by HW Mueller
Because pH is one parameter, organic concentration another?
Posted: Thu Feb 28, 2008 7:32 pm
by Bryan Evans
Does the protein precipitate when it is diluted in 0.1% TFA?
Or does the column clog when running the gradient?
They are 2 different issues.
Posted: Sun Mar 02, 2008 11:23 am
by danko
Good points Bryan!
Actually, it is not that difficult or time consuming to investigate these issues. And I usually do some simple testing before loading protein. Mixing the protein in question with the eluent intended for elution in a test tube will be my first thought (this advise has been popping up many times before in this forum and although quite simple at first sight, it is worth gold). In order to investigate your second thought (very relevant in protein/ACN context) it would be advisable to prepare 3 tubes with 3 different ACN concentrations: the first corresponding to initial conditions, the second corresponding to the ACN concentration in the middle of the gradient and the third corresponding to the end of the gradient. All 3 tubes should contain the same TFA concentration though. The next and the last step is, to add some of the protein to all 3 tubes and look for potential precipitations. It may take some minutes, so patience is advisable too
Best Regards