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Protein precipitates upon dialysis


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#1 research_freak

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Posted 30 March 2010 - 09:41 AM

Guys,

I'm trying to purify my GST-tagged protein. My protein is about 55 kD in size, combined with GST which is 26 kD results in 81kD of my GST-tagged protein. It forms inclusion bodies, so I tried to solubilize it in 8M Urea. Next , I tried to dialyze it in PBS, however, my protein precipitates.

There are only 5 cysteine residues and I doubt if there are disulfide bonds. Any idea, why the protein precipitates?

#2 K.B.

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Posted 30 March 2010 - 02:16 PM

What are you dialysing against? Water? PBS? Other buffer? Some proteins precipitate in too high or too low salt concentration.

#3 Denny

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Posted 31 March 2010 - 04:21 PM

Salt concentration and glycerol can be a major factor here. If your protein is stable in the elution buffer for a night in the 4C, Set up a series of buffers and dialyze on a small scale - Using a razor blade and 1.5ml plastic tubes, I just cut off the majority of the bottom of the tube. This leaves me with the snap top and the upper portion of the tube connected in one piece. Open the cap and place upside down, fill the cap with about 20ul of protein solution, cut a small piece of dialysis tubing big enough to cover the bottom of the cap, wet it in buffer and lay the dialysis membrane over the protein filled cap, snap what's left (1/8 - 1/4"max) of the tube bottom tube and place right side up in 100 ml of cold dialysis buffer overnight at 4C. By morning you will see cloudy or clear and hopefully find the right combination. I would, at minimum, try different salt concentrations and add glycerol to 5% and 10%.

Also, some proteins do not like the slow buffer exchange, if you have kits for quick buffer exchange, you could try a small amount that way. This is done by centrifugation.

I add a glycerol to 10% with unstable proteins upon elution or 5% in my all my buffers if I my purification process will allow it, and often have buffer with glycerol in the fraction collector of choice to:
1) dilute a highly concentrated protein quickly upon elution- many are unstable at higher concentrations (I've seen several that crash over 1mg/ml)
2) to stabilize with glycerol.

Just to cover all bases, everything that comes in contact with your protein should be cold. All buffers should be cold, unstable protein solutions need to be kept cold and on ice. ICE ICE ICE

Also note, that if the protein is misbehaving now, it's probably going to give you fits if you have to cut the GST tag off later.

Good luck, it's hopefully just a matter of finding the right conditions for your particular protein.



What are you dialysing against? Water? PBS? Other buffer? Some proteins precipitate in too high or too low salt concentration.



#4 Denny

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Posted 01 April 2010 - 03:25 PM

Also, just for fun, check the pI of your protein and make sure the pH of your buffers is right for the pI.
You could also try different pH in your search for optimum conditions.

#5 research_freak

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Posted 19 April 2010 - 12:56 PM

Also, just for fun, check the pI of your protein and make sure the pH of your buffers is right for the pI.
You could also try different pH in your search for optimum conditions.


Denny,

Thanks so much!! Actually, I'm trying to purify a novel protein that was discovered in our lab. Expasy has a tool that characterizes proteins for its stability based on its amino acid sequence. It characterizes our protein as being unstable. Also, the theoretical pI of our protein is reported to be 8.40. I've heard that there are different programs to calculate theoretical pIs of proteins. What are the other programs that could help in identifying the theoretical pI?

#6 ProteinWork

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Posted 19 April 2010 - 01:15 PM

Also, just for fun, check the pI of your protein and make sure the pH of your buffers is right for the pI.
You could also try different pH in your search for optimum conditions.


Denny,

Thanks so much!! Actually, I'm trying to purify a novel protein that was discovered in our lab. Expasy has a tool that characterizes proteins for its stability based on its amino acid sequence. It characterizes our protein as being unstable. Also, the theoretical pI of our protein is reported to be 8.40. I've heard that there are different programs to calculate theoretical pIs of proteins. What are the other programs that could help in identifying the theoretical pI?

I've been using this online tool: http://www.scripps.e...m/protcalc.html

But I think the theoretical pIs should not vary a lot from program to program.




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