Even a 15% resolving (non gradient should work). Higher MW might be due to post translational modifications. If you have a knockout negative control you can be more sure if the band is non specific.

histones -17 kda ,  i am rergularly doing 12% gel bands are good if u want u can do 12-13%.

Hi Bharat, I tried with negative control and I think these bands are specific. Post-translational modification might be a reason. I'm bit confused with the gradient gel. My ladder size is 250 to 10 kda. Since my bands are supposed to be resolved at the bottom of the membrane, I am just not sure to try with a 13-15% gel instead of a 4-20% gradient gel. Thanks for your suggestions.

Yes, I normally use Tris-Glycin-SDS or Tris-HEPES-SDS

Hi there,

I am working with 14kDa protein and 4-20% gels as well as 15% and 18% gels work fine for me. However, one thing that I noticed is that my protein band does not run consistently against the protein ladder (10-250kDa) depending on the gel percentage that I use. More usually it runs at a lower size but to a different extent on each gel percentage.

Nevertheless, if you are using a proper negative and positive control for your western blots you can be certain that your protein is being expressed. One trick that I use if I am in doubt is that I put a tag on the N- and/or the C- terminus that can be specifically detected with an antibody.

Maybe if you give us a little bit more information (like expression host and extend of secondary structure in the protein) we can suggest more tests.

Thanks Nicolas for your answer.

This is a tumor suppressor protein that is well-conserved across mammals. I am working with canine cancer models. But the antibodies I am using are for human as no dog specific antibodies are available. This is why I have run Western for both human and canine cell lines. I am also using a canine cell line that was transfected with human cDNA to express this protein. I have yet to finish with some more cell lines that never express the mRNA. 

1) Use a different ladder/make sure yours isn't expired.

LMW-SDS: http://www.piercenet.com/product/spectra-multicolor-low-range-protein-ladder

2) As Santosh said, use a 12% gel - preferentially in MES running buffer. I routinely use this setup to resolve 10-15kDa proteins - you won't run the protein of interest off the gel. If you're really worried, increase the crosslinker concentration in your gel to lower the pore size. 4-20% gels are typically used for electrophoresis of very small polypeptide fragments, sometimes in a Tris-Tricine system. Also, proteins are harder to transfer from higher percentage gels.

I generally use 12% gel and works fine for me for similar molecular weight protein. What kind of ladder are you using, sometimes prestained ladder shows different migration, it is more to do with the ladder rather than modifications, try using non pre-stained ladder  along with pre-stained if you really want to be sure about the size of the protein. I would not worry if I have specific band.

My ladder is just new. But I will probably try to use 12% gel.

Hi Aditi,

I am using Bio-rad pre-stained dual core ladder. I may try unstained to check the different migration of bands. - Thanks 

Using Tricin SDS PAGE (12 % separating, 4% stacking gel) I am able to detect 10 kDa proteins without comigration with the front. The position of 25 kDa protein marker (BioRad  dual color ladder) is approximately in the middle of the separating gel and protein bands are sharp after immunodetection both polyclonal and monoclonal anitbodies.

Hi Furruk,

There are two things I think you should check.

Proteins do not always run at their predicted sizes.  For example a protein that I was working with, alpha-synuclein which is 14KDa runs at approximately 17KDa.  Have a check in the literature for western blots of your protein to see what size to expect.  Companies often put in size and predicted size on their spec sheets.

Also, what ladder sizes are you using to compare your band against.  From my experience it can be hard to judge by eye the molecular weight of low molecular weight proteins on these gradient gels.  In our lab for example, we would compare your protein to the 15 and 25KDa bands, but due to the migration pattern 17KDa might be closer to the 25KDa than you might think.  I would suggest using a program such as imagej to calculate accurately the size of your band.

As always, pictures always help to diagnose problems, so if you can you should upload an image of you blot with the ladder overlayed.

Good luck with your westerns.

Cheers

Jacob

Thanks Jacob.

I think probably you are right and I am also assuming the same thing.I checked all literature and the companies spec sheets, and have not found the approx. band size on gel. I tried 250-10 Kd ladder. This is why I am going to run in 12% gel and small mol. weight ladder next. The pic will be uploaded with more info.