One possibility is that the 6his Tag on your receptor protein is unavailable to interact with Histrap. If possible please change the location of your 6his tag by cloning again and try out the purification. 

Another method is to denature the protein with urea and purify and then renature it again. 

I agree with Nagarjun's comments and would like to add one more. There is a chance that your His-tag was cleaved from your protein because of proteolytic degradation (you always need to use Proteolytic Inhibitor Cocktail). To test if this is the case, you might want to run your E. coli extract in western blot under reduced conditions and develop it with your anti-His antibody to see if the His-tag of your receptor protein will be accessible under completely denatured conditions.

If the protein is in a soluble, aggregated state, it may not bind well to the Ni column because the tag is not accessible. Purifying the denatured protein, as already suggested by Nagarjun Kasaraneni, is a way around this, but it is troublesome to have to refold the protein. Changing the expression conditions and/or the expression strain can sometimes help produce a more soluble, unaggregated protein.

Thanks for the suggestions.

I did added proteinase inhibitor during sonification and the result of purification is still the same which really troubled me. I am thinking of changing expression system.

Can I get some information about construction. Is the receptor protein Eukaryotic or prokaryotic, what about signal peptide. Is His tag at N terminal or C terminal. The transmembrane domains are generally hydrophobic, which may result in masking of tag. Further these hydrophobic domains tend to enter the membrane fractions. Changing expression system may not help. try removing trasmembrane domai, if it is not required for your experiments.

All the best

Hi Panduranga,

My receptor protein is eukaryotic protein with signal peptide on N terminal of the sequence. His tag is constructed on C terminal of vector. Transmembrane domain might be the main cause as you said.

Is the signal peptide for secretion by E. coli, or is it the natural signal peptide? Where do you find the protein - inside the cells or in the medium?

If you are expressing a full-length transmembrane eukaryotic protein in E. coli, it is probably just forming an aggregate or precipitate in the cytoplasm. Soluble aggregates often will not bind to columns. As Panduranga Rao said, if you are going to try to express it as a soluble protein in E. coli, you need a construct that does not have the transmembrane domain or natural signal sequence, just the cytoplasmic domain.

Hi Adam,

Its natural signal peptide from the protein, the expressed protein is located in the medium. What happened in my case is after sonication, i got most of my protein in soluble form, however purification through histrap column(plasmid constructed with 6xhis tag) is not working at all. I am thinking of adding detergent as i come across transmembrane domain might able to mask it

If the expressed protein was secreted into the medium, what was the purpose of sonication?

Hi Tang, as Victor asked, have you done a western blot of your protein to see if it still has the his tag after expression?

Adam, i can just centrifuge my bacterial culture and collect the supernatant. right? i never try that method before.

Steingrimur, i havent done that yet, i will try it, thanks for the suggestion.

If the protein is secreted into the medium, then you could collect it from the medium. The obvious way to begin is to pellet the bacteria, but if the protein is in a highly aggregated state, it may also pellet, so use the lowest speed you can to pellet the bacteria selectively. Of course, this does nothing to solve the problem of the protein not binding to the HisTrap column.