I am considering increasing the salt concentration, adding DTT, and including glycerol -- or some combination lowering the ph of my buffer. But little bit hesitating because I have to use it further for crystallisation purpose. Any other suggestions to prevent precipitation? Please reply

Mainly - decrease temperature!!! That should be the first thing to do.

Second, there will always be some precipitation in dialysis, because it takes so long. That's why people try to avoid it if possible.

Lastly, what are the buffers you use? The one in which is your protein and the one against which you dialize? If the difference is too big, that could cause denaturation. Maybe try subsequent dialysis.

thanks for your reply.the elution buffer 20mM tris ph 7, 100mM NaCl, 250mM imidazole pH 7 in which my protein and i dailysed my protein against 20mM tris ph7 having 100m M Nacl in it.

You can try to exchange buffer gradiently using concentrators. Although its its not good method but for some proteins it works better as they are very sensitive for dialysis.

Mam, do u knw the sequence of ur protein?? Means if it has cysteines then you better use any reducing agent during dialysis... Or else trying thermoflour assay will be other good option

The precipitation may be because of temperature your protein exposed during long time o/n. may be you can try dialysing at 4 degree as most protein are stable inthis temperature. you can use centrifugal dialyser and concentrator whcih take within an hour to dialyse and concentrate. you can try this at 20 degree and even if it ppt then do at 4 degree. i would recomment lower temp during dialysis as i dont see any problem with buffer. But i have one experience using tris buffer that protein precipitate. Is it possible to change the buffer and you can use that for crystallization? If you have higher volume you can go for magnetic stirring cell dialysis and concentration.

@ ashish I have 1 cysteine in my sequence....yep i m also considering to include reducing agent but one problem i've to crystalise that protein in its native form...........

thanks B. clarke and Arfi thanks for your suggestion i will do that one also :)

for sure you should dialyse at at least 10 °C, better 4 °C ... DTT could be a good choice as well... sometimes increase of salt conc. helps aswells.... temperature and salt are the main characters you should change.

Maybe you could try a desalting column instead of dialysis

Desalting column at 4C is the best. It is faster and reduese the oxidation amount. Same time you can increse the salt concentration, also.

if you are doing dialysis for the buffer exchange only then you cna dilute your protein elute with dialysis buffer and then try further dialysis. My experience says that imidazole can cause aggregation of protein. So diluting imidazole can help

Please check whether the initial pH of the buffer is 8.5 and also the pH of the protein precipitate in the dialysis bag. The strength of the buffer may be increased. Sometimes to the buffer used for protein extraction either DTT or glutathione (GSH) or cystine can be added. Dialysis may be carried out 5* C. This might solve your protein precipitate problem during dialysis

As well as high salt and low temperature you could try adding sucrose. I had the same problem and got over it by including 1M sucrose in my buffer

I think you should change pH buffer, and if you dialysis after ammonium sulfate precipitate you have to try reduce salt concentrate step by step. For instance your protein precipitate at 60% ammonium sulfate you should dialysis with 300 mM NaCl than change buffer with low salt concentration 100 mM NaCl. Temperature is important and I think that you should conduct dialysis in 4*C.

Hi Anamika,

I realize that this was an old post, but I am wondering if you were able to solve the precipitation issue after dialysis? I am facing the same problem and would appreciate your response.. Thank you!