Hi Ivan,

according to my experience, if you remove at the same time GuHCl and Triton, you have huge risk of precipitation if the protein is not properly folded.
In my opinion, you have to do something like re-folding. It seems that your protein could be solubilized from inclusion-body. In the same case, the first thing that i will do :
I start with the same protocol as you do, but after the TALON, i will dialysis gently the sample against the same buffer without the GuHCL but with Triton. Maybe you ca try to change the pH. Tris-HCl is a good strating point, but maybe you a to refine this. Phosphate buffer (7.5) helped me sometimes. What is the Pi of your protein ?

Hope to help you.

Nicolas
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De : CCP4 bulletin board [[log in to unmask]] de la part de CHAVAS Leonard [[log in to unmask]]
Envoyé : vendredi 9 janvier 2015 01:41
À : [log in to unmask]
Objet : Re: [ccp4bb] Protein precipitating at higher concentration!!

Hi Ivan

this will not be an answer to your question, but did you consider not concentrating 'too much' your sample? It happened few times to me that the protein was precipitating when concentrating for SEC because of the presence of other impurities. Trying the good old AS precipitation helped a bit, but wasn't really the magical solution as I was losing a bit of the protein of interest as well. The solution was to concentrate only slightly the sample, and pass it though multiple (at the time it was quite a lot actually) runs of SEC. I ended up with again a lot of pure sample to concentrate, however, this sample was pure enough and did not precipitate.

Other than that, I guess playing with the salt concentration might help keeping things stable... or not. I know people also tried the addition of glycerol or EG, but I don't have personal experience in that and cannot really comment if it is working well or not.

Cheers, Leo

> On Jan 9, 2015, at 9:00 AM, xaravich ivan <[log in to unmask]> wrote:
>
> Hi Xtallographers,
> I have been able to purify a protein that was initially going into inclusion bodies from the excellent suggestions that I got here.
>
> So my lysis buffer has 0.5M Guanidium Hydrochoride, 2% TritonX-100, 500mM NaCl, 5% Glycerol in 20 mM Tris-HCL pH 8.0
>
> The problem is that the protein is first purified as a SUMO-fusion protein which is further proteolysed and passed through the Talon resin to get the final SUMO-Free construct.
>
> However as I have around 250mM Imidazole (pH elution did not work) from the elution of the first round, I have to dialyse the sample to get rid of the imidazole so that I can use the proteolysed sample again on the column.
>
> All these I do in a buffer that does not have GuHCL or Triton. However I have kept the NaCl concentration same (0.5 M).
> I start to see white insoluble precipitate right from the dialysis step. If I spin the precipitate out, I still have a lot of protein to go to the next step of proteolysis. The problem is that when I finally want to concentrate the protein to run the SEC step, all my protein starts precipitating starting from 5mg/ml to all the way to 25-30 mg/ml.
>
> Are there some smart ways to keep the protein soluble at higher concentrations, assuming that I do not have to remove them before setting up trays?
>
> Should I keep on using Guanidium Hcl and Triton for all the steps all the way into the SEC column.
>
> Have people got any good results using 5% Acetronitrile, 50mM Arginine or DTT? (used for NMR samples)
> Any help in this regard will be very helpful.
>
> The protein is an engineered bacterial transcription factor. (not a membrane protein)
>
> Thanks in advance as always,
> ivan