Hello Scientists,
I am working on assaying the half life of a plasma membranal protein which is transiently transfected into Hek293T cell line using FuGene-HD transfection reagent.
Kindly, if anyone has experience using such measures, please do share your recommended protocol for a successful result.
Also, what could be the reason behind an increase in protein level despite the treatment? this happened with me on time-point 12 h of CHX treatment and falls again to gradual low levels till 24h of treatment. After 24h there is no increase in protein level.
B-actin is the loading control and its even for all lanes.
Nesreen Fayyad Al-Khuffash First of all do confirm that the dosage of CHX you are using, generally a 20-50ug/ml should suffice (then try increasing it). Do standardize the same with your cell line using a protein with a known half-life. In spite of this if the same trend is seen for your protein of interest, then maybe the half-life may be greater than 24hrs, though rare it is a possibility(Do check half-life for proteins similar to yours). If this is the case you can do a CHX-chase beyond 24hrs and depend on total protein staining for normalization than B-actin. Hope this helps. Good luck with your experiments.
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