I guess you try to purify recombinant protein from bacterial inclusion bodies. Right? In this case your are confronted with the magic arts of protein re-folding. Just trying differnt buffers may not be straight forward. You may need a more systematic approach. The best recommendation I can give is: (1) do literature search (if not already done) to find out what other groups tried with a related protein. (2) Get into contact with an experienced protein chemist as may require complex analytics to monitor proper refolding. I assume your protein should not only be soluble. In the end it should be functional as well.

Crystalline proteins are not very soluble in water. use a low ionic strength electrolyte to dissolve your protein.

i had dialysed three different proteins. One has dissolved completely. The other two did not dissolve completely.

After dialysis I had lyophilized the samples to reduce the volume. Will lyophilisation in anyway interfere with the solubility of the proteins?

It may. Meanwhile, the three proteins may have different solubility properties. Their solubilities may be affected by pH as their pI will be different. Play around ionic strength and pH of the medium in which you dissolve them.

You may have to change your dialysis buffer gradually and screen the initial protein concentration as well.

Not all the proteins dissolve completely in buffers and some times after dialysis, proteins may precpitate at the bottom.

It happens in case of highly glycosylated proteins as well, so if your protein is glycosylated , it's solubility in the buffer being used is being affected .In which case lyophyllization wouldn't help. Slowly change the ionic strength of buffer and it's pH ,one by one and optimize the buffer to be used for dialysis.

Do one thing ...elute the .Protein in 20mM Tris, pH 7.9, 500 mm Na-Cl and 1M imidazole. Another protein, size 32kD is eluted in same buffer but with 250 mM imidazole...if the ionic strength of electrolyte should be low....check the kinetic behavior of electrolyte.......check the pH level,,,,calibration should be there.......

Yes it happend

Depends on the pH of the buffer used for dialysis.

The buffer's pH shall stay away from the pI of your protein duriing dialysis. Adding some detergent may help keeping the protein in solution.

In my experience sometimes protein precipitation happens when their concentration is too elevated, above all after urea resolubilization. I prefer rapid disolution protocol, better than dialysis. pH and buffer concentration are also hot points.

make urea gradient i.e. if your protein is in 8M urea , then make 7M , 6M, 5M, 4M, 3M , 2M, 1M and finally dialyze it with the buffer without urea, u can add protein stabilizer in it like up to 50% glycerol and 500mM NaCl , instead of doing overnight dialysis.

I have similar problem with my protein purified via (native)IMAC, after my IMAC purification, when I try to concentrate eluates, considerable amount of protein start aggregates!

I tried different pH, salt, buffer, dialysis of course, but nothing seems to help my protein from aggregation :(

do you have some suggestion, I can also assume it may be due to "concentration dependent aggregation" with the contaminant protein(those 70kDa junks). but I dont know...

@ Ananda Ayyappan...if aggregation is due to contaminant, then better you go for second round of purification with e. g. gel filtration(size exclusion) or ion exchange chromatography, again i suggest add some protein stabilizers, add glycerol and NaCl or some detergents- Triton X-100 or CHAPS, and also many company like sigma ,pierce , quigen , provide kits which are having combination of diffrent buffers pH and detergents and also they provide protein stabilizers (they dont provide composition ), generally protein aggregates because of hydrobhobic region just go through this paper it might help you in designing your experiments link address is -http://www.ncbi.nlm.nih.gov/pmc/articles/PMC103993/.................................All the best...

Protein precipitation during dialysis against phosphate or Tris buffers (PBS or TBS) can be often caused by:

too low salt concentration (NaCl, KCl)

too high protein concentration

sudden pH changes

pH of dialysis buffer close to PI of a protein

Therefore:

use at least 1000 fold excess of dialysis buffer

perform procedure at 4c(at least 3 hours, followed by a buffer change)

use at least 350 mM salt in the dialysis buffer

optional: 50 % glycerol

Precipitated proteins are very difficult to refold. If possible purify your protein again and apply other conditions for dialysis

Dear friends..i do have same issue of precipitation with my protein, post Ni-His NTA purification..i am tried to solve this issue with using different pH of buffer (Tris),i even use mere distilled water as dialysis medium, adding 10% glycerol throughout during purification,however the issue still remain as it is, did not solve. I think, the precipitation is might be due to higher amount of salt (Imidazole or urea) which get abundant in the dialysis bag ultimately. plz correct me , if my consideration is incorrect. so would changing buffer after purification solve this issue? but i do not have any prior experience how should i change the buffer..? if somebody has idea, please do extend to me..Thanks..Besides,, i make a note of all the above valuable suggestions. i would work on it. Hope something work out..Cheers..:)

Precipitation of 6his-tagged proteins during dyalisis is a common inconvenient especially if the protein is concentrated or if, for various reasons, it results unsoluble, in the final buffer. In most of the cases I have solved this problem adding increasing glycerol concentrations in the dialysis buffer. My typical protocol includes 3 completely changes of buffer: 10% glycerol in the first change, 25% glycerol in the second one and 50% glycerol in the latter change o. n. You can add glycerol to the most commonly used buffers like PBS, TBS and so on... You can store the recombinant protein at -20°C without risk of degradation and use it directly for many purposes like immunization of mice or enzymatic reactions.

Dear Fabio Tosini..Thank u so much for ur valuable sugestion..i would certainly follow ur worked protocol...

@Ishrad Baig, Ananda Ayyappan Jaguva Vasudevan

Are you working with a soluble protein? I have found that passage through a dialysis buffer containing 100 mM EDTA can help prevent precipitation for some proteins. It seems to be something to do with Ni leached off the column, but I haven't done any formal analysis - it just works! The second dialysis should be your final buffer.

To change buffers during dialysis is very simple - just (carefully!) remove your tubing from the first buffer, and place it into a second beaker with fresh buffer!

Alternatively, run a desalting column instead of dialyzing. It seems to work better for some proteins.

@Richard Heath, Thanks dear Dr.Richard Heath for your kind response to my posted query.Yea Sir, i do work with soluble protein. I do use EDTA not 100mM but 5mM.,but the problem of precipitation still persist. my dialysis buffer is 20 mMTris,5 mM EDTA,5mM, betamercaptoethanol. besides, i have added 10-30% glycerol and switched over to PBS buffer, however these solutions does not work out in my case.

I do follow your suggestion,i would try running protein through desalting column. hope to get rid of this issue soon.

Thanks for your time and precious suggestions.

I guess you try to purify recombinant protein from bacterial inclusion bodies. Right? In this case your are confronted with the magic arts of protein re-folding. Just trying differnt buffers may not be straight forward. You may need a more systematic approach. The best recommendation I can give is: (1) do literature search (if not already done) to find out what other groups tried with a related protein. (2) Get into contact with an experienced protein chemist as may require complex analytics to monitor proper refolding. I assume your protein should not only be soluble. In the end it should be functional as well.

Brocks suggestion are right on!

you need to do dialysis against 10 times diluted buffer in which your protein is dissolved. Instead of dialysis you can try passing your precipitated protein through Sephadex G-25 column for removal of ions.

I do agree with Bodo Brocks 100%, urea is one of the agent used to un-fold recombinant protein from bacterial inclusion bodies prior to purification. Dialyzing to remove the urea right after a nickle column elution certainly will cause the protein to misfold and precipitate. In this case, I don't think changing the buffer/buffers will help bringing your protein back into solution. If possible, try to bring the urea concentration back ( to 6M or what ever molarity at which your protein was in solution) to unfold the protein then follow a proper re-folding method to re-fold your protein. Good luck

Thank you friends.

As suggested, I raised the urea conc. to 0.5 M and found the proteins resolubilizing.

Water is not a very good solvent for proteins. They dissolve better in solutions of electrolytes. Try salt solutions to dissolve the precipitate.

I had the same problem... may u can use either ion-exchange or gel filtration

Some proteins do precipitate on dialysis as they lose the ions which hold them together

increase salt concentration (na2so4 in place of nacl), or add glycerol (upto 20%), change pH of the buffer, away from its pI.

Yes, I also got precipitation of my protein after dialysis. Then i have added 500mM urea, then it was solubilized.

Proteins are more soluble in solutions of ionic salts than in distilled water. An interplay of the ionic strength of the medium in which proteins dissolve can help us to precipitate or keep proteins in solution. That is, "salting in" and "salting out". Since dialysis helps to remote small ions and proteins below the cut off of the dialysis pores, the resultant effect usually is decreased solubility of the retained protein, hence the usual precipitation of such proteins after dialysis.

..remove..." not remote, please. Thank you

The precipitated protein was dissolved by addition of 500mM UREA. Can i use the dissolved sample for further purification by gel filtration. It will help to remove the retained urea (500mM) from sample. Can you suggest that may again get precipitate after removal of urea during gel filtration. Thanks

Hello, I have a cuestion: I have a His-tag protein but it precipitate after the Ni-NTA purification, I need to concentrate with a centicon... It is posible do this?

Hi Cleotilde,

You can use, You have to select the right column size based on your desired MW of protein. This step not only concentrate your protein and also able to remove co-purificants.

Cheers.......