Hi Chandni

Dialysis is a slow method for buffer exchange. Some proteins have time to get into weird unstable conformations and precipitate. To avoid that, I will suggest you to use a faster method for buffer exchange like PD-10 columns.

Another thing is that when you dialyzed you removed two important things: ionic strength (PBS has around 150 mM of Na and K ions, but in your initial purification buffer you had 300 mM) and glycerol. I will also suggest to increase a little bit the NaCl in your final buffer and also to include some glycerol. Proteins typically need some ions to get into solution.

Good luck. Best

Paco

Hi Chandni

Dialysis is a slow method for buffer exchange. Some proteins have time to get into weird unstable conformations and precipitate. To avoid that, I will suggest you to use a faster method for buffer exchange like PD-10 columns.

Another thing is that when you dialyzed you removed two important things: ionic strength (PBS has around 150 mM of Na and K ions, but in your initial purification buffer you had 300 mM) and glycerol. I will also suggest to increase a little bit the NaCl in your final buffer and also to include some glycerol. Proteins typically need some ions to get into solution.

Good luck. Best

Paco

Thanks Francisco. Next time i will try and add glycerol but what percentage should i use for this as initially i was using 10% and for Nacl what amount should i increase to maintain ion in solution.

Chandni...

You can use the same percentages that you had in the elution buffer, so 300 mM NaCla and 5-10% glycerol. By the way, your pI sounds very high... is this a DNA or RNA-binding protein?. If yes, use a little bit more of salt for the dialysis (500 mM), because those guys love the salt.

Good luck

Francisco, its secretory protein then in this case too i should add 500mM Nacl? 

I suggest you to try step dialysis and its an effective way when we purify the protein in pellet fraction in the urea. What we follow is the allow a slow change of buffer with reducing concentraions of urea every 2-2.5 hours ( 4M, 2M, 1M and 0.5 M and then i PBS overnight rather than putting in PBS directly. In this way you allow the protein to refold properly. One important point to consider is to elute in higher volume than usual. You can concentrate the protein with centricon later. Hope it helps good luck

A none P buffer sound like the right way, but I would beg to differ regarding the dialysis. Dialysis does not have to be very slow, I tested once and found that 35S-Methionine is distributed equally after 15 minutes, this was done at room temperature with a stirrer in the outer beaker. Using of the more modern dialysis slides/micro would probably make it even faster. A big advantage of the dialysis over ultra-filtration is the the later elevate the protein concentration dramatically and some proteins tend to precipitate. Furthermore, some proteins bind to the membrane irreversibly, although pretreatment of the membrane with BSA should help.

Bottom end, try

Hi

I suggest you to remove imidazole first, and then reduce salt content by dialysis or desalting on PD10 column.

Hi, 

I agree with others, during dialysis you should keep your protein in the same conditions, except for imidazole, like in elution buffer as you know your protein likes it (you were able to purify it and it was stable in the elution buffer), because eliminating of glycerol and dropping down the concentration of salt (both important) is too big stress for your protein. Also, you should continue using beta-ME/DTT if you didn't. I wouldn't suggest going through Amicon ultrafiltration units, as imidazole is not going well through the membrane and you will introduce even more unknown factors (like, you don't know how much you can concentrate your protein etc. and you can easily over concentrate it in these units). You can also try to lower the concentration of imidazole in your elution buffer, if you didn't, down to 300mM or 250 mM, maybe you don't need 500 mM?  You can also use FPLC columns like Source S as your pI is high to eliminate imidazole and further purify your protein, but in this case you need to check first on the small sample if your protein can survive lower concentration of salt, without changing other variables. Anyway, you can try a few things on a small scale until you find best conditions.   

Good luck.

Hi Chandni,

1. Try to dilute the Ni-NTA fractions immediately after elution (at least 1:2) with your buffer (or even water, if your protein will tolerate the drop in salt concentration) before trying to buffer exchange.

2. Test different buffer-pH/salt combo to figure out the more stable conditions for your protein. Importantly, if it is not a thermostable protein, try and keep it at 4C. 

3. You may already know this - many contaminating E. coli proteins that co-purify from Ni-NTA type method tend to crash out when buffer exchanging.  Therefore, it is important to know that the protein one is trying to purify is actually the protein of interest.

Best wishes.

Thanks everyone. I will try some of your suggestion.

If your protein have free cysteines (not forming disulfide bonds) be sure that you have reducing agent in your dialysis buffer. DTT has short halflife, especially if buffers are not degassed. B-me-ethanol in 4C, pH7.6 is stable for few days.

I am not sure if anyone mentioned it. My friend met similar problem with antibody fragments. They observed precipitation during dialysis or after few days if kept in 4C or after defrosting. It was caused by histag interactions. Histidines (and not only) can bind bivalent ions that are present in solution (impure water or any other ingredients). Via histidine and ions protein in solution can bind to each other and cause precipitation.

To get rid of these ions you may use EDTA 0,5-1mM or 5mM imidazole if these aren't non compatible with your experiments. EDTA also stabilizes you reducing agent and tailors its halflife (also bivalent ions influence). EDIT: sory, you had imidazole in buffer:)

I hope it will help you.

Best regards

Try step dialysis and try to elute the protein in larger volume of buffer.

if u will reduce the concentration of protein or either reduce buffer concentration or both concentration the precipitation will not occur during dialysis

Hi Chandni, a good option to preserve protein solubility in native conditions is to add arginine. Add arginine to final concentration of 0.2 M or higher to your dialysis buffer. In my hands arginine made protein not only soluble but really stable (it significantly enchanced prolonged storage at +4C without any decrease in immunoactivity). 

Actually I have to use this protein for interaction so any modification can alter interaction In that case what should be done.

It doesn't seem like anyone has mentioned this, but after eluting your protein from the Ni-NTA column try to dialyze it with a buffer containing 0.1M EDTA. (the first buffer I used was 0.1M HEPES, 0.5M NaCl, 0.1M EDTA) then switch to a second buffer to remove the EDTA (the second buffer I used was 0.1M HEPES, 0.5M NaCl, 10% glycerol). I had a similar problem with my protein precipitating after Ni-NTA elution and the EDTA helped a lot. The EDTA helps to chelate the leached nickel from the column. Hope this helps.