Hi all,
I am working with an intracellular domain of a type1 transmembrane protein. This intracellular domain is really small (9 Kd) and contains a predicted NLS. I made two constructs (with two different expression vector) of this ICD with and without its NLS sequence (I just deleted the first 9 amino acid of the ICD) to understand the functionality of that NLS.
I performed western blot to check the expression of my constructs.
Every time I am getting nice band from the ICD from both vector but not from the ICD w/o NLS constructs.
I generally run 4-12% bis-gradient gel and blot for 2 hours at 200mA with whole cell lysate.
Is it something to do with my western blot protocol or my ICD w/o NLS is not really stable?
p.s: I have done ICC also and there I got nice stained cells from this ICD w/o NLS constructs and apparently the staining spared the nucleus.
If anybody has any expertise with low MW protein blotting or with NLS constructs, please share your experience.
Thanks.
You don't mention what part of the construct was used to raise the antibody, and if it is monoclonal or polyclonal. Is it possible that it is a polyclonal and the antibody that reacts with denatured protein is directed against the NLS sequence motif, but that reaction against a native determinant shows up by immunocytochemistry?
Hard to say but i suspect that ICD is degraded. Proteins that don't find themselves to correct location are often degraded. Try general proteasome/protease inhibitor (ie MG132) to see if it prevents degradation. Your ICC may be nonspecific staining.
alternatively,the deletion of 9aa may have changed binding characteristics of your protein. Could try different blot material - PDF or nitrocellose. Also your transfer is excessive for such a small protein.
David... I use a polyclonal antibody and your point is quite right I think. Thanks for this idea!
Gary.. Thanks for your reply! I normally use protease inhibitor I was also doubtful about transfer time .. will try to reduce it definitely!
Just to clarify; It may be degraded within the cell during the expression. Incubate MG with cells during expression. You must use cell permeant inhibitor.
Did you count the percentage of transfected cells in your ICC? If you have a low percentage of transfected cells probably you are under the detection threshold in WB. Why you don't try to load a nuclei enriched extract?
Giovanni,
Thanks for your reply.
I thought of that but the percentage of transfected cells in ICC was okay.
But anyway, I will do WB with nuclear fraction now.
I usually use 16.5% Tris-Tricine gels and their specific cathode and anode buffers to separate small proteins/peptides. You can get a really nice resolution if you pour them between large plates with thin spacers. But apparently your current gels seem good enough to see your protein. Did you also try PVDF membranes with small pores (0.2 µm instead of standard 0.45 µm) that are improved for the transfer of small peptides ? For some small proteins, I also immerse the membrane in boiling PBS and allow it to cool down before blocking and it actually improves the detection. I hope it may help you.
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