Hi, I ran western blot with two proteins with increasing concentration from 5 ug to 25 ug ; b-actin(control, 42kDA, lane 2-5), and Foxm1(protein of interest, 100kDA, lane 6-10).
They were from one lysis from cells, and ran on same gel (7.5% TGX gel from Biorad), blot(0.45um nitrocelluose), transfer buffer(standard Tris/Glycin buffer with 20% methanol) at the same time. Only difference was the primary antibodies to detect each protein. However, both antibodies were purchased from the same company at the same time, and they were not expired yet.
Do you have any idea what would cause this issue?
It is very frustrating since it was run on one gel and all the condition has been same.
Thanks,
Since there are 3 isoforms of FOX M1, perhaps the diffuse band results from the detection of all 3 isoforms with slightly different mobilities in SDS-PAGE. Also, varying levels of phosphorylation could contribute to the diffuseness of the band. (https://www.uniprot.org/uniprot/Q08050#ptm_processing)
I agree with Dr Shapiro. There are 3 isoforms of FOX M1. I don't know the company where you get your primary antibodies from, but CST showed that the protein will be expressed in isoforms as shown in their official website (https://www.cellsignal.com/products/primary-antibodies/phospho-foxm1-thr600-d9m6g-rabbit-mab/14655). The isoforms are also published in Nature (Figure 2, DOI: 10.1038/NCHEM.1114).
Thank you so much Dr Shapiro and Dr Rhaman,
I would have never thought of FOXM1 isoforms contributing to this phenomena by myself.
I appreciate it.
Best,
Bumjun
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