Purified Protein Aggregation/Multimerization Problem - (Nov/15/2006 )

Hi, I'm a graduate student who is new to these forums.

I have been purifying a DNA binding fragment of the transactivator I am interested in. I have a His-tag on it and am purifying it from BL21 cells. I am using a double purification system, using both a nickel column and a heparin column, yielding a very pure prep at high concentration (2-5 mg/ml).


After some initial odd results in my DNA binding experiments, I looked into the state of my protein and found through native gel anaylsis that it seems to be multimerizing (giving me a ladder of bands).


I store my protein with the His-tag still attached in the following buffer:

50% Glycerol
100mM NaCl
10mM Tris-HCl pH 7.5
5mM EDTA
.5mM DTT

@-20C


The protein should be in a natural dimer state, with a predicted pI of ~10. Since I don't see a smear at all in the native gel, I'm unsure if this is actually aggregation. Any insight into what conditions might help resolve this problem would be appreciated.

I have initially tried increasing NaCl concentrations from 100mM-1M with no real effect to speak of on the multimerization.

Thanks in advance.

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you could try increasing the dtt to as much as 2mM.

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There is only 1 cysteine residue in the protein, would DTT still make much of a difference?

Thanks for the quick response.

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welcome to the club!

there seems to be a lack in renaturation; try to optimize renaturation, it is not to underestimate; there are some forum debates about renaturation; use the search mask


for future questions, please try to be more closer to the point although a detailled question may help to understand a more complicated problem

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