You did not say what the SDS-PAGE looked like after the 1st affinity purification step. Were there multiple proteins detected then? If the answer is yes, then your situation does not seem surprising or unusual. It is just that the purification method was not completely effective at removing impurities. I think you should add an additional chromatography step, namely ion-exchange, to improve the purity.

oftentimes, minor impurities co purify with your protein after affinity chromatography and sizing columns are typically as effective as ion exchange as Adam has mentioned. But you can use the sizing column or other desalting procedures such as dialysis to put your protein in a low salt buffer and then try cation or anion exchange. You can also try hydrophobic chromatography such as phenyl sepharose, or if your protein is glycosylated, I would try first ConA or what germ agglutinin. For phenyl sepharose, you put the protein on the column in high salt and then use a high to low salt gradient to elite. For ion exchange, you go from low to high salt to elite.

Dear Adam and Marcia

Thanks for you reply, the problem is not with affinity chromatography because already I know it is not pure, so I tried gel filtration which should separate between differen molecule sizs, I mean when I collect one single peak I found it my protein bound with other three proteins and my question is how to unbound these three protein? then when I run gel filtration I have to get four clear peaks, 73kd, 47kd, 48kd and 125kd. so the last one is my protein which can be collect sparately form others

Which gel foltration column did you use? Did you calibrate the column with known proteins, this would also help you understand how close to gether proteins will elute. Do you see your protein of interest in all fractions, 700, 400 and 250 kDa?

Maybe your target protein was not properly expressed. It could be miss-folded or even aggregated. The expression strategy is also important. 

In the absence of greater detail about exactly what these contaminants are, it is only possible to make some general comments on possible reasons and solutions:

1. The larger aggregates (>250 kDa)

These could be co-purifying with your protein for several reasons

- they bind non-specifically to your affinity matrix. If you are using IMAC, increase imidazole in your binding and wash buffer to 20-40 mM. If it is an antibody affinity column decrease pH slightly. Other affinity methods such as GST or MBP - check the manufacturers' troubleshooting guides.

- They are E. coli proteins that are binding to your protein of interest. In this case try buffers with different salt concentrations and different pHs (in the range of a couple of points above and below the pI of the protein you want). We routinely use 0.5 M NaCl in our lysis and wash buffers but test a range to optimise for your protein. Changing pH and ionic strength can help to dissociate protein complexes - but take care that you don't create new ones!

2. The smaller bands (

Many Thanks Deepan

Already I did what do you suggest in number one, just I did not tell you that I run the protein on SDS gel then I cut the single band which is my protein and sent it to mass spectrometer but the result is like this mixture.

 Thanks all for your answers, I collected the peak which in may be my protein, and I run this on SDS gel then I cut the single band which approximately  125kd, but the Mass spect result give this mysterious thing.   

which coloumn do u use for GFC?

Hi Sneba

HiLoad, superdex200

Hi Hadi

I think you should not directly go for GFC in this case. try 2 steps of IMAC. Make sure that your Protein is clean enough. Keep 2 different tags on your Protein to ensure purity after 2 steps of IMAC purification. (Like combination of His & Strep).

Once you are assured that you have got rid of all the impurities, try GFC for clearing out the rest. Dont go for SDS PAGE, rather go for native PAGE if you are not sure of the oligomeric assembly of your Protein.

Good luck!

Sneha

Thanks Sneha for these suggestions, actually, it seems to me that there are three proteins bound strongly to my protein becuase it still remain bound during gel filtration so I think these non specific proteins are not exist as a contaminant in the protein solution but in abound state to my protein.

thats what! if you do stringent ourification of your Protein These intracting ones might get flush during the second IMAC.

if at all it doesnt go away, try Ion Exchange chr. Or try to figure out if These can be potent interacting Partners physiologically as well.

Maybe your protein was complexed with these two  additional proteins? ( If affinity chromatography was in more or less native condition) So on SDS page YOU CAN SEE THEM

Thanks Wojciech, exactly yes I can see them on the SDS-PAGE

Hi Hadi,

In order to get rid of protein impurities you can use ion exchange chromatography. The protein sample will bind on the ion exchanger (eg. Q column) and then you will use a titration of salt to remove the proteins bound through electrostatic interactions. Αfterwards, SDS-PAGE will show you the optimal salt concentration to collect your protein. 

Thanks Dimitra for your answer

Sounds like there could be some E. coli chaperones in your preps if you haven't found the answer by now. I would read into things like getting rid of DnaK, for example. My best guess would be that playing with your wash steps at the affinity purification stage may be the most efficient. Options would be to add glycerol or mild denaturants like 2M urea (make fresh) or so to those buffers once your protein is bound to the beads. May have to disrupt hydrophobic interactions between chaperones and your target protein so high salt washes may not do the trick.