Hi all.
I am a PhD student who work in protein purification and crystallization. A few months ago I started with a protein which molecular weight is very big around 100kDa (monomer). When I purify this protein I obtain in the Superose6 column 3 peaks with a Ve of 8ml, 9,4ml and 10,5ml ( this Ve to PM is 2MDa-1MDa) but the relation between the intensity of the peaks changes from a purification to another (For example : In one purification the 8ml peak is the biggest but in other purification the biggest is the 10,5ml peak). In the SDS-PAGE I see a contamination: DNAk that I cant separate it from my protein ( DNAk is more concentrated where there are more protein). I proved a lot of additives: L-arginine, L-arginine+glutamic, glucose, saccharose, some detergents, glycerol... And only detergents change something: I saw with DLS that the monomer appear in the graphyc when I add 0,2% of Tween-20 in the sample of Superose 6, but In a low concentration.
I proved the ultracentrifugation method with a concentrate of Superose6 samples (the same I obtain before) and the result was one peak in a PM: 100KDa(monomer).
I dont know If the peaks are a oligomer mix or It has some relation with the DNAk but I want/wish obtain the monomer or stabilize one of the oligomers. Any suggestions?
If you have some questions I will try to answer it.
Thank you.
When you did the ultracentrifugation experiment and saw monomer, did you include Tween-20?
Was there any effect of protein concentration on the amount of monomer or size distribution?
Have you used any other chromatography steps besides gel filtration to try to separate DnaK from your protein?
1) No, I didnt use Tween-20 in the ultracentrifugation experiment ( Hepes 50mM, 200mM NaCl and 25mM Saccharose)
2) No, the concentration hasnt got effect in the structural conformation. I proved differents protein concentration and the results are the same.
3) My purification steps are HisTrap (Ni column)- MonoQ (anion exchanger)- and Superose 6( Size exclusion).
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