I'm trying to determine the half life of the protein that is expressed from the plasmid and has auto degradation loop. So supposedly it should degrade faster than its non-loop counterpart. I've done some reading about cycloheximide, Is it possible to do this with non radioactive method, and can this be applicable in this instance with cell transfection? Can the half life be determined by arresting the cell cycle in G0/G1 phase and performing the western?
Determining protein half-lives.
Zhou P1.
http://www.ncbi.nlm.nih.gov/pubmed/15173609
hi..I hope this link might be helpful...
The reference by Rishi can provide the details concerning acceptable approaches to determine the half-life of a protein of interest in cultured cells. However, the method involving cycloheximide (at an appropriate concentration), which inhibits new protein synthesis, could have an effect on the half-life of a protein (because turn-over of certain proteins requires the protein synthesis). Therefore, pulse-chase experiment involving labeling protein with labeled amino acid followed by a chase by unlabeled amino acid may provide better results.
We have several publications describing that. For studies of turnover of unlabeled proteins, 5 million cells were treated with 100 μg/mL cycloheximide before harvest, whereafter cell lysates were immunoprecipitated . se figur 2 in this paper:
http://www.bloodjournal.org/content/95/6/2104.long?sso-checked=true
Thank you, would i still use Actin or GAPDH as a control, will it be influenced by cycloheximide
You might want to also probe your blots for p53 or something similar with a short half-life to act as a positive control, to check the CHX has worked.
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