I am detecting concentration of IL-6 in U266 cell line by western blotting. The result showed two blots, how could I solve this problem?
Do you mean the result is showing two bands? If so, you can contact the vendor of the manufacturer of the antibody. Ask them for the peptide or protein that was used as the immunogen. You can try incubating the immunogen (in excess) with your antibody and see if one of the bands go away. Hope that helps.
Hi, Yangping, have you used poly or monoclonal antibody? Does band have same intensity? Does at least one band have MW similar as you expected? In the case if one band is very low and far from expected size, you can try to optimize incubation conditions. Good luck!
i THINK problem may be with your protein u need to check before western that your protein is not degrading or getting cleaved, may BE some how like by RNase but in this case intensity will be different , higher intensity will be target .....
It may be helpful if you can show us the blots and detail what kind of antibody you used (poly or monoclonal). Also, some ILs share subunits or share homology which may give you multiple bands on WB.
Thank you for your kind respond. The day before yesterday was Chinese new year which we call spring festival, so I wish all of you have a happy new year.
I read the manual of the IL-6 antibody, found that antibody is polyclone, it could detect 21-28kda IL-6.Maybe that`s why I observed two bands.
I enclosed my result below.
The bands are too close together to make a difference of 7kDa. I assume you ran a standard 10% SDSPAGE. I think the bands differ with only 1kDa. This is not enough difference to be explained by glycosylation. You should compare this result with a different IL6 antibody. One of the two bands may not be IL6.
Once, I've read that partly oxidation could lead double bands.I do not know in detail but why i could reason that the differences between the bands are not that high. Increasing the amounts of b-mercapto in sample buffer in order to prevent this modification on the protein is offered in that text. I dont know if this could work and have not experienced this solution, but could be tried easily. By the way, did u found an answer as it seems much has took from last answer. Good luck.
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