I am purifying a recombinant protein (host Rosetta) with his tag. I am getting reasonably good yield (4-5 mg/liter of culture). However after affinity purification using Ni-NTA column, during concentration protein is precipitating. I am using Amicon 10 KDCO centricons for concentration and I could observe that even at 0.4mg/ml concentration protein is getting precipitated. Protein buffer is 50 mM tris pH 7.5, 500mM NaCl, 5% glycerol and 10mM beta ME and pI is ~6.0. Protein is eluting at 250mM Imidazole, is imidazole causing the precipitation?
The precipitation is as a result of lower solubility of the protein at higher concentration. In order to increase solubility at higher concentration there are many ways like increasing salt concentration, adding osmolytes like glycerol etc.If the size of protein is larger or it is a protein complex then aggregation is likely to happen. Also check whether your protein is hydrophobic or hydrophilic. The membrane for concentrating has to be selected accordingly .
good luck
Hi! Manjula,
This problem which you are facing is common problem for me. My protein used to precipitated during concentrate process. The fault is in imidazole. You better dialyzes your protein in appropriate buffer overnight (change the buffer 3-4 times in few hours gap). You can add 5% glycerol as well. Then concentrate on centricons or vivaspin. Decrease the concentration of NaCl while dialysis.Sometimes high salt concentration may occur precipitation depends on the nature of protein.
Hope this work for you.
All the best.
Higher pH (8-10) might sometimes help for solubilizing a protein, if your protein allows it. Think carefully about high salt concentration, because on the other hand high ionic strength increases hydrophobic interactions which may lead to protein aggregation. I always do the dialysis right after Ni-NTA column.
Good luck!
imidazole concentration was removed after affinity and that worked fine but after gel filtration (20mM tris-Hcl and 150mM Nacl) its precipitating in corning concentrator. Need help.
Hello! You could try a large PD desalting column. Bio-Rad and GE sell them. The spin one from GE is nice, and quick. You could prepare the desalting column before elution. Elute, dilute, desalt. Then when you concentrate with the amicon you'll pretty much know if your precipitation is concentration dependant. You may need detergent. There are a number of things to look at on the protein level. Good luck!!
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