I am working on Epithelial to Mesenchymal Transition (EMT) in A549 cell line by treating positive inducers such as EGF to up-regulate the mesenchymal markers like vimentin. I'm doing western blot to detect the changes of vimentin expression level over treatment time of the inducers. I could detect the protein band of vimentin at the position of its proper size, but the change of protein expression level flutuate too much.
For example, I expected increase of the vimentin expression level by the treatment of EGF at least in early timepoints right after the treatment, but it wasn't. Neither at longer imepoints. Sometimes the level of vimentin was completely disappear, sometimes increased, or just remained in the same level. I always confirmed the expression level of beta-actin or GAPDH as a loading control, and it was consistent.
I tried same kind of expreriments using mesenchymal cell line like MDA-MB-231, and in this case, the degree of the fluctuation was decreased but it doesn't completely disappear.
The passage of the cell lines are all around 10, which is I think low enough, and I grow my cells in complete medium RPMI 1640 (10% FBS, 1% Penicillin/Streptomycin) and I treated the EMT inducers after 16 hours serum starvation.
I doubt that the A549 cells that I prepared for the experiments were not in good condition, so this can be the reason of the fluctuation. Do you think that the health of the cell can affect to the level of vimentin much? If so, how can I troubleshoot my problem? Any suggestion or advices can help me a lot. Thanks in advance.
The problem is with gel concentration and high molecular weight of Vimentin. Use 8% gel and try to load 40ug protein instead 20ug. I hope you will get it. I have worked with EMT and got Vimentin blot in my master thesis. A549 I think it is NSLC cell line. Our previous lab has done , no problem with cells as well. Good luck.
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