u shud add NaCl 150-250mM in your elution buffer. do not adjust pH for imidazole, use 200mM imidazole. Try to change the whole buffer system to Tris. rest depends on ur protein's activity. Generally addition of salts stabilize the proteins, and do not harm their activity. For more details of the protocols, u can follow our papers, as this is the routine job for us.

if the pH of your buffer is too close from the pHi of your protein of interest it can precipitate. what is the pH of your buffer? and the pHi of your protein ?? sometimes, precipitation occurs after protein concentration and this might be due to some problems during protein purification. if you can give me more details maybe i can help you cheers

Dear Gregory

At first thank you for your help. I am using pH 7.4 25nM K2HPO4 buffer solution. Theoretical pI value of protein is 6.60.

Protein did not get precipitated before the concentration and desalting steps even up to 15 days at 4C , but it is getting precipitate during/ after concentration when I used the K2HPO4, Tris-HCl (pH=7.4 )buffers and it stable when I used the DI water.

first you can increase just a bit the pH of your buffer as usually one pH unit of difference is required to avoid precipitation. Also, you can check your pH during each steps of protein purification, if it goes less than one unit of difference this could be sufficient to induce aggregation of your protein during concentration. you can put some methione and arginine in your lysis buffer avoid oxydation and favor folding of your protein. if there is some hydrophobic patch in your protein, maybe you have to add detergens to avoid precipitations. what have you already try ??

I haven't add any of detergents and additives, because I want to study the activity assay under pure protein condition. Now I would fallow your suggestions too, please can you suggest me what are the additives/ detergents should I use to avoid the denature and precipitation of the protein. I am basically from Maths and Chemistry background in UG and Graduation level, but I have chosen Chemical Biology as a my research career. So, facing some troubles, but suggestions from experts like you can be very helpful in my research.

Thank you time and consideration.

first check the pH if you would like to add some detergents add some FC12 which is really good

Hi Srivardhan. Use this website and check if you're preparing your buffer correctly http://www.liv.ac.uk/buffers/buffercalc.html

The other thing which you should try is use pH around 8.0 rather than 7.4. Ideally you should use 1 pH unit away from the pI of your protein as Gregory Berger said in one of the previous comment. If your theoretical pI is 6.6 then best to use pH 7.6 or more i.e. around 8.0. Also check if there are any cysteine residues in your protein sequence as they may result in aggregation due to disulphide linkage. To prevent aggregation caused by disulphide linkage you might need to add a reducing agent to your buffer.

Dear Sunny Singh and Dear Gregory Berger

Thank you very much for your time and consideration.

I would follow of your suggestions

in fact i do not know how it work but by adding methionine in your lysis buffer you will prevent disulphide linkage between two cysteine and arginine favor fold ing of your protein. the rule is you have to protect your protein from oxydation and aggregation from the beginning of your experiment (lysis) alwais check the pH (one pH unit of difference between the phI and the pH of your buffer). if you have any problem let us know good luck

Dear Gregory Berger

Thank you, I would try this procedure, I will let you know if when I face the problem.

u shud add NaCl 150-250mM in your elution buffer. do not adjust pH for imidazole, use 200mM imidazole. Try to change the whole buffer system to Tris. rest depends on ur protein's activity. Generally addition of salts stabilize the proteins, and do not harm their activity. For more details of the protocols, u can follow our papers, as this is the routine job for us.

I agree with Andaleeb. I too had 150mM NaCl in my buffer when I was doing Affinity Nickel column chromatography. Also storing the protein in Tris is recommended for long term storage if you want to keep in -80 deg. C. Usually if the protein is in Phosphate buffer and if you thaw/defrost your protein from -80 deg C then some of it does crash out. So it is safe to work with Tris.

Dear Andaleeb Sajid, Dear Gael Cristofari, and Dear Sunny Singh

Thank you very much for suggestions and your discussion on my problem, I learnt a lot from this discussion.

Hope now got confidence to work on it, your people made my doubts clear. This discussion really very helpful for new researchers like me. Again thank u all people for your time and consideration.

http://antibody-antibodies.com/search_full.php?search=nvoy

You need nvoy its a sugar wich enhances hydrohilicity of yoru potein, it has lyphophilic binding capacity and has a hydrophilic side...

I have doubt here. Imidazole pH value. It was what? Is it not about 10 and more? First test the imidazole flakes pH after dissolution. Did U add HCl to adjust pH? Imidazoles nature?

Dear Thyaga Raju Kedam

I haven't checked the pH of Imidazole also haven't added HCl. I have repeated whole experiment and I would follow the all suggestions whatever mentioned here.

Thank u sir, for your information

Dear All

Thank you very much for your kind information and suggestions, at finally I could solved my problem.

Hi did it work finally? Waht did you change ?