protein precipitation during concentration - (Jul/15/2006 )

Hi everyone

I'm a new entree into this forum. I read the topics dicussed in the forum and it seems very interesting and useful for me. Many of them I do on the benchtop, but some I didnt know what it does...thanks..really really useful !!!!

Anyways I would appreciate if anyone could give me some suggestions regarding protein precipiation during concentration.
Expressed as 6xHis-tag protein in pET28a, the theoretical pI is 7.9 and Mol wt. 30kDa. The histag is at the N-termi. It binds to the Ni-NTA column and elutes at 250mM Imidazole when i do a linear gradient. On the Gel-filtration (S75), first time I used 10mM Tris-HCl, ph8, 500mMNaCl, 1mM EDTA. I used Amicon concentrators (10kDa) to concentrate, but the protein precipitated .
The 2nd time i used 50mM MES pH6.5, 500mMNaCl, 0.1mM EDTA (for S-75)--- precipitated.

Also I centrifuged and collected the supernatant and then concentrated, it precipitates again. I tried to resuspend the pellet to get back the protein...but no luck.

Could anyone give me some tips. ALL kinds of suggestions are welcome.

Thanks

Sukumar

I have spent much time struggling with the same issue earlier this spring. I found that for my protein, I have to use neurotic conditions to get native purification and still maintain solubility. As per the Qiaexpressionist and my boss' prior experience, glycerol was added to lysis/elute/wash/dialysis buffers to 10%, triton-x 100 to 0.1%, and NaCl to 2M (this was all added to the NaPhoshpate/imidazole buffers recommended by Qiagen) All steps after sonication performed at 25C, with buffers pre-equilibrated to 25C, even the centrifugation to clear the lysate. Protein stored at 4C until extracted, purified, dialyzed, and assayed; then aliquots were taken and stored at -80. Fortunately, my protein is biologically active when diluted quite far in cell culture media, so that I was able to get away with this.

Good luck

Since i'm going to crystallize the protein, i think 2M NaCl is little too high. I probably think to use 1M salt with 5% glycerol in all my buffers.

Thanks for the valuable suggestion

Sukumar

Just a suggestion: you may also try lower salt concentration. High salt promotes hydrophobic interaction.

Hi
Such a coincidence.
The same thing happened to me last week.
In a paper was saying that my protein could be concentrated up to 30mg protein /ml.
So I used vivaspin columns to concentrate protein. Protein precipitated!
Lucky me that I didn't transfer all the protein to the column so I still had a lot of protein.

What I understood is that with these type of columns the concentration of the protein closer to the membrane can be much higher than in the top of the tube/column therefore, I think close to the membrane the concentrtion of protein exceeded the 30mg/ml and precipiated but there was still a lot of protein soluble in the upper part.

I didn't try yet but I know there are are methods to concentrate protein which make use of a filter and have a kind of magnet constantly rotating the protein sample to make sure concentration is the same in all sample.

Yes, I donn't know for crystalagraphy but I think you are using too much salt.
Before concetrating sample you can desalt protein and substitute buffer to a bufffer with 150mM salt.

Good luck,

Hi everyone

Using 5%glycerol in all my buffers didnt help much still protein precipitated. Also tried to used another method to concentrate using the constant magnet rotating method..still no use.

Since the protein has 6XHis at the N-terminal, will it help anyway by cutting the Histag?
OR
do Dialysis/ desalting column to reduce/completely remove salt.

Note: I need to crystallized the protein so some amount of salt is needed for crystallization.