My protein is His-tagged and has a molecular weight of 27 KDa. I expressed it in Rosetta E.Coli cell. I used a 1 litre culture to extract soluble protein. After purification using Ni-NTA column I ended up with multiple bands along with the protein of interest.
IMAC purification was done as follows:
The colum was equilibrated with 2 column volumes (20 ml)
Equilibration buffer: 50mM Tris-HCl, 100mM NaCl, pH 8.0.
Then the protein with Ni-NTA agarose flowed through the column.
Then washed with wash buffer 1 (50mM Tris-HCl, 100mM NaCl, 20mM imidazole pH 8.0) twice.
wash buffer 2 (50mM Tris-HCl, 100mM NaCl, 40mM imidazole pH 8.0) once.
Elute buffer 1 (50mM Tris-HCl, 100mM NaCl, 100mM imidazole pH 8.0) once 1ml.
Elute buffer 2 (50mM Tris-HCl, 100mM NaCl, 250mM imidazole pH 8.0) 4 times 1 ml each.
I understand number of washing steps and imidazole concentration helps to remove contaminated proteins. But in the washing steps there is almost no proteins. 100 mM has some in the elution 1. and in elution 2 there are multiple bands. So 250 mM works for both my proteins and the contaminated proteins.
I have attached a picture of my gel run. I use DTT in the loading buffer during running SDS-PAGE gel. From L-R; flow-trough, wash1-1st flow, wash1-2nd flow, wash 2, elute 1, elute2-1st flow, elute2-2nd flow, elute2-3rd flow, elute2-4th flow, ladder.
I appreciate any help in this matter.
Edited by Bioscientist, 16 January 2013 - 10:08 AM.