Hello all I apolozize for a off topic question on BB.
I am working with small proetin-protein complex, each have molecular weight 10kDa. I elute this N-terminal His-tagged complex through Ni-NTA resin in 50mM of NaH2Po4, 0.3M of NaCl, 5% glycerol and 250mM of Imidazole.Similarly, Lysis buffer and wash buffers contains same component without and with 20mM of imidazole respectively. His tag is present on the N-terminus of one partner of the complex. I am able to purify this protein in small scale and eluted fraction contains 1-2mg/ml of protein complex. But, when i scale up the purification volume to 1-2 lit of culture i get a really good yield like 1or 2 fraction will have 10mg/ml of proetin complex.I store the eluted fraction both at 4 degree as well as 25 degree. The fraction contains higher amount proteins get precipitated and settles on the bootom of microfuge tube while the fractions with less amount of proetin(1-2mg/ml) remain soluble. can anyone suggest any way to keep this protein complex solube without breaking the complex? Whether there is any other way to screen the solubility of protein with different buffers without using dynamic light scattrer? please suggest any reference which suggest protocols to check the solubility. I would appreciate the help. Thanks in advance Peter
