Let me explain my problem, after a good yield of ionic exchange chromatografy (used as a second step of purification) my protein that should bind to another protein expressed at the same time, is degraded. But is this degradation due to protease still present, or the solution may be to use different buffers to stabilize the complex and proteins?
Assuming you're using a protease inhibitor cocktail in your lysis buffer (without EDTA of course...Ni column), and you're running your columns under refrigeration, then I doubt that your problem is protease degradation after two steps of purification. What is your evidence that your target protein has degraded?
Yes, of course I used protease inhibitors (sigma cocktail EDTA FREE, PSMF (2mM) and Benzamide)...the evidence is that after ionic exchange I had my 2 bands of my two proteins on SDS page analysis gel, but after gel filtration I lost my protein of 15kDa...any suggestion?
Do you have an estimate of the purity and amount of target protein you have prior to loading the GF column? How much are you losing? Also, what are your GF conditions? Type of column, etc? Your protein is showing activity in your assay prior to this third purification step?
Dear Daniela,
Proteins in complex should be protected rather, than promoted for protease digestion, thus I find your problem as an really unusual... My initial ideas are:
- your column is contaminated by proteases, becomes active in the mixture (10% chance)
- the E. coli protease co-purify with your protein, but their inhibitors go into another fraction, at the time of complex reconstitution it becomes active due to the environment change (15% chance)
- your second interacting protein is contaminated by the proteinase is digesting only the first one... (20% chance)
- moon phase (55% chance;)
Harsh column wash with NaOH/urea/HCl at the max conc. recommended by the producer will not disturb your exp. anyway. Any protease activity assay of your mixed crudes of two proteins can give you a clue, but I'm not in your situation, so this is rather suggestion...
Keep fingers cross - Jan
Let's say...sorry for my very few wxplanations...but I'd like to remedy...I expressed my 2 protein with pETDuet system in BL21. It has been demostrated that these two proteins interact, I would like to obtain a very pure complex to crystallographic studies. My problem is that after my ionic exchange cromatography
I had e good amount of proteins...in the file (first lane induction, second and third lane NI-column purification, then thare are the eluition fractions of ionic exchange...It's seems a good yield...but after the third step of purification I lost one of band...(one of protein)...how it can happen?
Could be the salt conc. mentioned by Paul is destroying the complex. Your fractions loading is far from ideal, could be this is salt excess in the samples. Anyway the fractions are still contaminated with the proteins of higher MW. No degradation is observed. I ddn't get what you mean by third step....
Well as you want to get rid of the impurities, use TALON resin instead of NiNTA. I crystallized my protein complex after co expression with pETDuet vector in Bl21 cells. I found a lot of difference in the two resin. TALON is better in a sense that it doesn't binds to many E.coli proteins and therefore you can omit the IE chromatography step and directly go for Gel filtration. If your protein is degradation prone try to do both Affinity and Gel filtration in the same day.
Good luck with your experiments!
generally high salt conc. interferes in better resolution. first you ensure that samples were stacked properly during electrophoresis. and run the sample after removing salt with membrane filter or dialysis. Second check your both proteins bind with Ni column because in your picture it's not seems like that.
Hi to everyone....only my first protein bound the resin because it is tagged with 6Xhis, the second protein bound my first protein and my purpose is purificate the complex
Dear Daniela,
Without the details indicated above it is very hard to conclude about your gel, where the lines are not described. Just my feeling is that your complex during IE is destroyed by increased salt, than the subunits bind again to the resin, eluting separately due to the to high salt at the end of the gradient... Pure speculations at this moment. Why not to change this not working strategy (both in theory and in practice) into the standard biochemical complex reconstitution. A variety of techniques like: size-exclusion, tricks with tagging both proteins, in vitro reconstitution of highly pure proteins; is available. With high amount of your crude proteins the easiest experiment to plan further strategy is to find the salt concentration range you can work with your complex without the fear it will be destroyed. For this purpose I would use or the Ni-bound proteins with increased salt gradient wash or size-exclusion with a set of columns equilibrated with different salt...
Best - Jan
Go with gel filtration immediately after nikel cromatography !!!!
Hi everyone, for Jan my third step of purification was gel filtration...and after this step my complex seems to be destroyed (I couldn't see one of protein on gel).
About the buffer that i used for ionic exchange here they are: for equilibration of the column i used 50mM Tris pH8; 200mM NaCl (plus protease inhibitors): then for elution i used 50mM Tris pH8 and 1M Na Cl; for gel filtration (the step in which i think i had trouble) i used 10mM Tris pH8,200mM NaCl plus 2mM TCEF
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