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Fail to see under physiological stimulation

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Fail to see under physiological stimulation

Postby kingsupercat » Wed Aug 21, 2013 2:48 am

Hi all,

I am working on a protein that had been proved to be phosphorylated on its Tyrosine in the presence of constitutively active Src mutant. However, I had been trying for an year to see if I could reproduce the result using PDGF to turn on endogenous Src activity and the results are all negative! Does anyone have good suggestion what to do next, or how to breakthru?

thanks a lot!!

samuel
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Postby JackBean » Wed Aug 21, 2013 1:39 pm

Change project? Burn all your notes?

Now seriously, have you confirmed the Src is active in your system?
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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Postby kingsupercat » Thu Aug 22, 2013 3:10 am

Haha JackBean, that's really funny.
And yes, I'd seen the Src being activated using pY416-Src antibody under the stimulation...
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Postby JackBean » Thu Aug 22, 2013 11:40 am

how was it constitutively active? What's the PGDF? Is your protein endogenous or does it has any tag?
http://www.biolib.cz/en/main/

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Postby kingsupercat » Mon Aug 26, 2013 2:37 am

It's a single amino acid mutant Src that makes it always on. PDGF is one of growth factor that is known to activate endogenous Src. I had tried looking at both GFP-tagged exogenous or endogenous but all fail to see the phosphorylation of my protein evoked by PDGF.
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Postby JackBean » Tue Aug 27, 2013 9:36 am

activate Src in sense of expression or phosphorylation? Can you check the Src is expressed in the first place?
http://www.biolib.cz/en/main/

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Postby kingsupercat » Wed Aug 28, 2013 4:34 am

yes i am sure it was expressed and i also saw the activation of Src under PDGF stimulation detecting with pY416-Src antibody
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