Trouble with Protein Elution

[vip5]

I am user of Ni-NTA beads and i am using it for purification of recombinant protein. I am facing a problem with the purification of my protein and i need your advice in it. Actually i am purifying a protein of 40 KD having a pI of 7.6, having 10 free cysteins when i purified it for the first time i used TRIS buffer pH 8.0 but the protein got precipitated. After that i used different buffer to elute it out of the beads.

Lysis Buffer:

50mM TRIS+ 500 mM NaCl + 10 mM Imidiazole + 20 mM Beta - mercaptoethanol pH 8.5

Washing Buffer


50mM TRIS+ 500 mM NaCl + 10 mM Imidiazole + 20 mM Beta - mercaptoethanol pH 8.5

Elution Buffer

50mM TRIS + 500mM NaCl + 250mM Imidiazole pH 8.5

This time i was able to elute it out but on doing dialysis it completely got
precipitated.

So can you please suggest me what conditions should i change to purify my protein.

One more thing when i used 20mM Beta-mercaptoethanol in elution buffer also it turned out to be red. But no such effect during binding and washing. I think it can not be due to beads reduction because after elution beads didnt turn brown. Can you please explain my query as i am suffering from past 2 weeks :-( :-(


Hope to hear suggestions soon :-) :-)

Vip5

[N.R.]

"on doing dialysis it completely got
precipitated." - maybe you need to change pH of the dialysis buffer to 6.0-6.5 for example? The isoelectric point of your protein is theoretical, maybe it's not 7.6 exactly and get closed to 8.0-8.5?