Help on native PAGE gel and its western blot result - (Dec/03/2014 )

Hello everyone, 

I need some help to interpret my native gel and its western blot. The image is shown below

(or here: http://postimg.org/image/9jtythz2h/)

Description of the image: 

The black and white image is the western blot x-ray result of the native gel (Coomassie staining) shown on the right. 

In the western blot image: Lane 1-6, 8-10 are the same protein sample. Lane 7 is a protein ladder. Two bands were bound to the anti-his tag antibody (indicated by the red arrows)

In the Coomassie gel: Lane 1-2 are the same protein sample as the western blot (two bands, indicated by the red arrows)

(I ran two native gels, one used for coomassie staining and one used for Western blot)

The protein sample is a secreted recombinant protein, btw it is a monomer. (no cell lysis step to harvest this recombinant protein and this recombinant protein is almost the only protein in the culture supernatant (>95%) )

So my questions are:

1. Because the western blot has lit up both bands on the gel, so I am confident that both those two bands are my protein because my protein is the only his-tagged protein in the supernatant. So how come my monomer protein got separated into two regions on the native PAGE gel? 

2. If both bands are the same protein, how come it showed two different migration patterns? And how come the top band (from the coomassie gel) is SO MUCH SHARPER than the lower band? What can be a possible cause?

That's about all my confusions regarding these results for now. Thanks for reading and helps are much much appreciated...!

1) No and yes - no in that if your antibody isn't used at the right concentration, or not washed enough, or the secondary incubation was too long or concentration was too high then you are likely to get non-specific binding. Yes in that if the above are all OK, then the bands should be His tagged bands.

2)Perhaps there are more than one folding states, or perhaps the protein dimerizes with another protein (not itself). The sharpness could be due a number of things, first to consider would be the size relative to the percentage gel concentration.

Thank you very much bob1!. I am pretty certain that I did wash thoroughly and didn't overkill the secondary anti-body concentration...Plus, the ladder wasn't bound to the anti-body at all (as seen in the x-ray pictures). So to me that's one thing suggesting I probably don't have non-specific binding. Secondly, I ran another native gel before with a negative control (supernatant from cells without my recombinant gene but induced in the same way as the cells with my recombinant gene), in that gel, I didn't see any protein bands on the negative ctrl lane...This is another reason why I am pretty certain both of the sharp band and the blurred region you see on the coomassie gel above are my protein (and most likely the same protein)...

So if the separation is due to different folding state like you suspected...could it be because my protein somehow got glycosylated differently? (my protein is a glyco-protein). What could be some possible ways to correct the folding problem? Thank you again in advance! 

Antibody not binding a ladder is no indication of specificity, most ladders wouldn't/shouldn't  bind antibodies. Non-specific binding can easily happen under any conditions and it is very important that you validate conditions that you use before you start any work that will give you a significant result. Another thing to check would be if the antibody is known to work on native proteins, most don't.

It could be glycosylation, phosphorylation or any other post-translational modification causing the size shift, but you would have to check charge structures and all sorts of things to determine this properly. One way you could validate is to cut the bands out and get them put through a mass-spectrometer (usually Orbitrap or MALDI-TOF-TOF).

I wasn't implying incorrect folding, just different folding states, perhaps a relaxed and active conformations, or perhaps presence of a cofactor (metal ion?) causing changes in folding state. Correct folding state may or may not be correctable, you can look through the literature and see if there are any crystal structures published, and look at the conditions used.

you may also be seeing the effect of proteases on your protein.

Thank you guys. Just wanna clarify some terms. So is relaxed state the same as denatured? or relaxed state = partially denatured? or relaxed state doesn't equal to denatured state at all?