half life of a protein (View forum version)



soymilk14

Posted 18 August 2013 - 11:12 PM

I just want to ask what are the simplest but publishable method on  how to determine the half life of a certain target protein without using radioactive assays. thank you so much. 


Inmost sun

Posted 19 August 2013 - 05:46 AM

half life depending on what conditions? isolated/purified, lyophylized, in vitro, in vivo?


soymilk14

Posted 19 August 2013 - 07:15 AM

oh thank you.. i just want to ask how can i know the half life of an endogenous protein in-vivo. thank you


bob1

Posted 19 August 2013 - 01:07 PM

The simplest method is to block the protein transcription with something like cycloheximide and harvest lysates at different time points, then run western blots for the specific protein.  Your band of interest should disappear with time, and the intensity can be measured to give you a rough half life.  It isn't very quantitative though, so accurate determination of the half life is difficult.