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Equal loading for Supernatant western blots???


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#1 bluedoozer

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Posted 17 July 2009 - 07:23 AM

Hello everybody.

Ive a interesting question for all experts in western blotting out there.
I entered recently a new group and for the first time in my life I need to make a western blot with supernatant.
So far I managed to preticipate my supernatant with ethanol to concentrate my amount of proteins!! thas great for the beginning

But when we run a western we also want ,as usual with a normal western with cell lysates, a equal loading control .

Does anyone knows a appropriate control for supernatant western blots.

Im also think of semi quantifiing my results with imagej and therefore i would need also a reference in form of a equal loading control!

Can anyone help me with this problem???

Thanks in advance

bluedoozer

#2 polyfractal

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Posted 22 July 2009 - 02:31 PM

"Housekeeping" proteins are often used. Pick a cytosolic protein that you don't feel is going to be affected by whatever problem you are studying. Classic example are Actin, Tubulin or Gapdh. It is also a good control to nanodrop your samples first, normalize protein quantity then load your gels based on the normalized values. This plus an adequate loading control will help immensely during quantization of your blot.




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