Here I have attached SDS-PAGE gel picture of Ni-NTA affinity chromatography elution profile of a protein at varying concentration of imidazole in tris buffer(pH 8). I have done washing in 10, 25 and 50mM imidazole concentrations and eluted at 400mM concentration. I can see a high concentration of my desired protein in 3rd collection (lane 8) tube but with some smears below.
What is this smear? Degraded protein or ghost band or contaminating protein?
I have run the gel at room temperature.
The extent of purification of your protein looks good to me. When you overload your gel with any homogeneous protein you usually see some minor bands like yours. I think your preparation as it is can be used for any structural or functional characterization. If you want more homogeneous preparation you might want to use a gel-filtration column and collect and pool only those parts of your major peak which look more homogeneous to you.
My guess would be contaminating protein. NiNTA and other Nickle columns don't give very clean products most of the time. They're great for a first column in a purification, but it's often necessary to use additional columns to further purify the desired protein. Another option would be to run a step or elution gradient to try and separate the desired protein from the possible contaminant.
That said, the protein you have purified looks rather good if the NiNTA is the first column in your purification. Your contaminating bands make up perhaps a few percent of the total protein detected in the gel. This may be good enough for some molecular biology applications.
If not, I've often had luck following NiTNA up with either Heparin or MonoQ. These columns are convenient because the buffers are vaguely compatible. You can dilute your NiNTA eluate in an appropriate salt-free buffer until the salt concentration is low enough to get binding to either Heparin or MonoQ.
The extent of purification of your protein looks good to me. When you overload your gel with any homogeneous protein you usually see some minor bands like yours. I think your preparation as it is can be used for any structural or functional characterization. If you want more homogeneous preparation you might want to use a gel-filtration column and collect and pool only those parts of your major peak which look more homogeneous to you.
The lower band may be a truncated version of your protein arising from proteolysis. A Western blot or mass spec sequencing will be able to show whether this is the case. If you are planning to try to crystallize the protein, it may (or may not) be necessary to improve the homogeneity of the preparation. For some uses, it may already be pure enough.
I am going to do enzyme kinetics study with the purified sample. I will take the last lane sample which looks better than others to avoid any confusion.
Tahnk you all for your valuable input.
The easy answer is a contaminant protein. From my experience it is almost imposible to get totally pure protein preparations. The contaminant could be a different protein, an interacting partner or a degradation product. would advice to do mass spectrometry of the bands if you want to nknow more.
The band spread looks like it might be owing to protein overloading. You could test this:-
Cut out the band and slice the gel into sections ( say 3 or 4) along the length of the gel so that each section of the band contains a portion of the band with a different mobility: that is from the leading edge to the trailing edge of the band. Incubate each gel slice separately in sample buffer so that that the SDS binds to the protein but insufficient time to elute the protein. I guess 30min but you might have to test this. Put each gel slice into the sample well of a new gel and rerun. Use a gel lane for some of the original sample as a control. Alternatively you can macerate each gel slice in sample buffer to elute the protein. Remove gel particles by centrifugation and use the supernatents as samples on a new gel. If you see only one band with the same mobility in each sample then all the protein in the original band has a single mobility.
Could be either overload or contamination.
Peter's test is right on and does not require any expensive equipment.
- Badges
- Science method