Hallo,

I have a problem with the purification of my His-tagged protein:
After the purification I can see additional bands in the gel, so I don't have 100% pure protein. I used to have no problems with that. I learned the purification not where I do it now, but I use the same buffers and do everything exactly the same way.

The additional bands I see are:
1. bands smaller than my protein
2. bands smaller than my protein that I can already see in the lysate
3. bands bigger than my protein I can see in the lysate
4. bands bigger than my protein which are at the very top of the gel (therefore must be very hugh)

1. could be due to degradation but it never happend before... All buffers are cooled in ice before usage so the room temperature shouldn't make a difference.
2. and 3. could be because some interaction or unspecific binding, but why didn't that happen before???
4. could be DNA/RNA????

I need to have pure protein for my assays so I need to remove all these additional bands!

A bit more information about how I do the purification:
I express my protein in E.coli DE3 BL-21 RPL and lyse them with the following buffer:
20 mM Tris; 500 mM NaCL; 10% Glycerol; 20 mM Imidazole; pH 8.0.
And I am also adding 1 mM PMSF, 2 mM mercaptoethanol, 1% Triton, Lysozyme and DNase.
I keep it stirring for 2 h at 4C and after centrifugation load it onto a Ni-NTA-column and wash with cold buffer before I elute it with cold buffer.

As I said, I do the protocol exactly like I learned it but now I get these additional bands and have no idea what I can do...

Hope you can help me. If you need to know anything else, just ask...
Thanks in advance.

I supposed that you see the bands in a SDS gel electhrophoresis so I think the bigger bands can no aggregatinos from your protein but can be glycosilated forms. wich kind of protein is? membrane? cytosolic? First at alll I were did a western blot of the sample with anti-his antiboy for see if the band have the his tag or not. Second, triton X-100 1% is too much and can solubilize too much non desireed proteins. try with 0.1%. After sample aplication wash wih 4 volumen of 50-100 mM imidazole. and after a gradient up to 500 mM.
good luck