i am working with a set of plasmid constructs for HCV proteins ; each one is separately loaded with individual FLAG-tagged protein ............ after transfecting the NS4B containing plasmid in my cell line and harvesting it i did a Western blot analysis and to my surprise i found that i had 2 bands ; one at the correct size "29 KD" and another one at about "55-65 KD " ........ i repeated it and i had the same result ................... Do anyone have an explanation for this findings ?
Dear Mohamed,
A few details we would need to know in order to help:
Did you use an antibody against NS4B or FLAG? Which antibody did you use? Which cell line are you using? Native or denaturing Western Blot?
Some antibodies are not very specific and may crossreact with other (similar) proteins (proteins often have domains and if antibody was made against a "domain" sequence then it may recognize more than one protein), some proteins dimerize (double the size could suggest this is the case), cell surface proteins often show distinct bands for partial or fully glycosylated forms....
A quick lit search gives this paper which may be helpful (but I've only had a quick look, so may not be)
http://jvi.asm.org/content/85/16/8158.full
Also aparently flavivirus NS4B dimerizes:
http://www.ncbi.nlm.nih.gov/pubmed/24390334
Did you titrate your antibody? Sometimes you can get additional unspecific bands... papers often only show partial blots, so you don't know if they too had additional bands.
Hi Mohamed,
As Petra said it really depends of your assay conditions and especially of your antibody. It is not uncommon to find 2 or more bands if your antibody is not specific enough or if it’s used at low dilutions.
NS5A protein can be found at two different phosphorylation conditions and indeed you can see two distinct bands on gel. However I don't think the same thing applies to NS4B.
If you are using Ab to NS4B my best guess is that second band is a non-specific one or maybe a protein-dimer?
Try to increase the antibody dilution and use more stringent conditions it may help you.
Best regards,
Bruno
If this is the non-specific band, it should also appear for the non-transfected cells. From the size, it seems like a dimer.
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