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-   -   What would a western blot with siRNA knockdown show? (http://www.molecularstation.com/forum/rnai-sirna-forum/83972-what-would-western-blot-sirna-knockdown-show.html)

Musicalrain 11-06-2011 09:16 PM

What would a western blot with siRNA knockdown show?
 
Say the protein of interest is cyclin D of the G1 cyclin-Cdk complex. What could a western blot utilizing siRNAs show? Thanks in advance!

Ted K 11-06-2011 09:16 PM

If you compare your siRNA-treated cells with control cells, you should see significantly lower expression of cycln D on your blot, i.e. load equal amounts of total protein when you run your gel, then if the siRNA worked, the band for cyclin D will not be as dark as the one for the controls.

Jon Moulton 11-08-2011 03:58 PM

Re: What would a western blot with siRNA knockdown show?
 
There will be a time lag between siRNA treatment and good knockdown. The siRNA will prevent most of the new translation, but preexisting protein will turn over at its usual rate. For some proteins, like many transcription factors, the turnover will be fairly rapid so you can see a good signal decrease within 24 hours. For others, like some structural proteins, the turnover might take several days to detectably decrease the signal on a Western.

Apoptosome 11-16-2011 04:20 AM

Re: What would a western blot with siRNA knockdown show?
 
Cyclin D is highly abundant, especially in cell cultures with ~60% confluence. I dont see an siRNA knockdown significantly affecting a western.


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