Purification through the Ni-NTa column - (Feb/28/2005 )

We have protein with size -30kDa including His6 tag .One of the problems is that we have small amount of protein after expression by using RTS system (ROCHE- Small - Scale Protein Expression). The total volume of the reaction mix is only 50 ml (micro L, i can not type -sorry)...we tried to purify through the Ni-NTA column but after elution still we have multi band . We have used purification under native conditions, We tried different concentration of imidazol into washing buffers )we started with 5mM than 7mN,10mM,20mM,40mM,60mM,100mM) Keep in mind that the conc. of imidazol in elution buffer is 250mM...After all purification steps the result is multi band...
How we can solve this problem,could you help us?
After Western (by using anti His6 peroxidase conjigate) we received only one single band...
Thanks in advance!

Ni-sepharose is know to give multiple band despite being an affinity matrix , when metal binding proteins are present in your sample. The alternative is to try with other fusions like GST. If you would like to go ahead with the present one itself then you will have to further purify the protein. The eluent from the Ni-sepharose column can be subjected to other chmromatographies like ion exchange, gel filtration, hydrophobic interaction............

good luck