Hi everyone,
I used anti-Egr-1 (rabbit monoclonal, ab133695, Abcam) in western blotting experiment and I see multiple bands ranging from 50kDa to 130kDa, where at 130kDa bands are more intense comparatively. I do not understand at what molecular weight I should find the actual band, it's predicted mol wt is 57kDa, but I do not see any band near to that. In literature it shows 62kDa and 80kDa.
Why there is a difference in the mol. weight?
Is it usual to get multiple bands in Egr-1 western blotting?
Thank you very much in advance!
Dear Amisha, may be Egr-1 exists in its multiple isoforms (splice variants), which is a common phenomenon in several genes. But, you need to keep in mind the other issues causing multiple bands of your POI. So, all is to do more & more troubleshoot. I am going to summarize some of the common issues in western blots related to multiple bands so you can figure out something at the end hopefully.
Multiple bands if are towards the low-molecular-weight region mostly indicates degradation (repeat your extraction carefully with some adjustments). Presence of any PTMs in your POI at the time of extraction may also result in multiple bands.
You should increase both primary and secondary antibodies dilutions to see this effect persists or appearing more better with higher dilutions, as many times only the appropriate antibody titration gives you properly placed single band.
One of the underlooked reason for multiple bands is to use a single protease inhibitor during extraction not the cocktail one.
Try to get cross-adsorbed antibodies if you can, as they are extra-purified hence decrease cross reactivity and enhance specificity by large.
Multiple bands are common if you use polyclonal antibodies.
You can also run an experiment without using the primary antibody and complete the run with your secondary and if still there are multiple bands, so problem is in your secondary antibody.
Loading too much lysate in gel causing antibodies to bind nonspecifically, especially to proteins of abundance or housekeeping proteins no matter your antibody is directed to which protein. Some of the epitopic configuration of HK proteins may match with your antibody.
Charged SDS molecules and other impurities of gel system and tissue lysate components (salts, buffers, debris) may also cause antibodies to bind nonspecifically. So if you can cleanup your lysate before loading onto gel it would be great. Following kit in this aspect if you can afford so it would be quite helpful;
https://www.thermofisher.com/order/catalog/product/89888
Regards.....
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