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How to concentrate conditioned medium for Western

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chanchunky

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 Send a personal messsage to chanchunky Reply with a quote from this post Go to the top of the page

Hi,
I am looking for some secreted proteins in the conditioned medium of cerebellar granule neurons. Since the amount can be small, I tried to use the Amicon Ultra-4 Centrifugal Filter Devices to concentrate the media before running them on SDS gel. But after doing that the gel would run funny - looks like partially dissolved. And I wasn't able to detect any clear bands. On the attached picture, the left 5 lanes are the CGN cell lysate, while the right side should be CM after concentration.
Does anyone also use such devices for the same purpose? Or would you suggest I try other means of concentrating the CM?

Thanks a whole bunch!



Attached file: Centrifugal Filter.jpg
Centrifugal Filter.jpg
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Posted Jun 29, 2007, 6:51 AM
gsovak

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Did you tried speed vacume concentration?
I guess that the problem that you had is that some other proteins or other lipids where also concentrated and thus the gel run funny.
Maybe the way that I sugested whould help.
You can always try to percipitate just proteins with TCA and clean the pellete with acetone.

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Posted Jul 04, 2007, 21:29 PM
chanchunky

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Thank you very much! I'll give the speed vac a try.

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Posted Jul 06, 2007, 1:26 AM
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