LC sample load & solubility advice? - Shimadzu prominence (Sep/19/2006 )

OK maybe this is a dumb question for all of you but I am curious as to any ideas. I'm purifying this protein that is not very soluble. I have used Ni chromatography already to begin purification but it needs a Q column to remove almost everything else.

I don't have any Q bulk material so I can't make my own column but I have a 1 ml and a 5mL prepacked that I have used on the next lab's Shimadzu prominence. I can't concentrate the protein much as it is only soluble up to 0.5mg/mL in detergent and I am purifying without even any detergent (I need it monomeric)!

I've used HPLC before many times but this has never come up, so don't call me stupid for asking. Can I load more than the max sample loop (5mL) either with multiple injections or by some sort of continuous loading method? I presume it would be ok because it never elutes in a very sharp peak, so it would come off the column dilute as well. I imagine I could load and then start the run to load protein and stop the run and repeat several times but what exactly will happen if you overload the sample loop all at once?

Alternatively can I set it up to continuously load by sucking sample out of a beaker or something, then switch to the normal solvents/gradient? I could keep it dilute this way and then the Q column usually removes unwanted proteins that limit my protein's solubility (so it should be ok after this).


Alternative dumb question if you don't mind.... what about running on another anion exchanger like DEAE or something (which I have in bulk to make my own columns)? Has anyone tried this? I may do an experiment with some of the sample on DEAE just to see how it looks.

Thanks for any help or ideas any HPLC users or experts out there may have.

yes, you can load more than the loop volume. do this by either continuous loading (but you will lose some of the sample in the tubing when you change over to your buffer or you will introduce bubbles into your column) or by multiple injections from the sample loop. do not overfill the loop, you will just send your sample to waste. i am assuming that the initial buffer conditions are conducive to binding of your protein and you are planning to elute your protein by changing buffer conditions (salt, pH, etc.) otherwise you won't be able to use your 5 ml loop.

you could use DEAE but remember that Q (QAE) is stronger and will bind the protein tighter.

hope this helps.

Ah, yes. Thank you very much for the advice. What I ended up doing was manually loading the Q HP column with a syringe and then running the gradient. This was because I wanted to dilute the sample in binding buffer (so should have been ~20mM salt, should come out at >100mM salt on gradient) and it is not a very soluble protein. However, then I ran into the problem of not being able to blank the run so I had negative absorbances and I had to quick manually collect the fractions!

Only problem was that I later found out from Western that none of the protein bound. It was in the flow through! Since it was a used column I decided to try again with a fresh column but I think it ran through again.

We're going to have to figure out what's going on but perhaps that could be why my last run failed too (we're trying the full length version of a protein - with it's hydrophobic tail group...when I tried this with the very soluble truncated version it worked fine! I tried full length with detergent and without and it wont bind the Q column either way!) Other groups have done this procedure before so I'm pretty sure it works, just not sure what's wrong this time.

Thanks a lot, anyone else have similar problems before?

it's possible that the pI of the full length (with hydrophobic tail) is significantly different from that of the truncated peptide. this could cause weaker to no binding to ion exchange. you can either try a range of pH buffers to find where it binds or try cm or sp ion exchanger (for cations).

No, the purification of full length and truncated both used anion exchangers (Q) when done before. I beleive the pI I have listed (a little above 5 for this human version) is for the full length and I do not know the exact pI of the truncated. I will double check all publications on the topic though and check the calculated pI again just to make sure.

Here, pI 4.9 according to http://pedant.gsf.de/cgi-bin/rep2.pl?Db=Mu...;Title=c7000981