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Western difficulty in primary neuron cells.


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#1 Fatsheep

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Posted 04 December 2010 - 10:07 AM

I did a western blot using lysate from cultured primary neurons, the results is very disappointing. As usual, b-actin gave very nice and strong signals, but all my target proteins such as Akt, phosp-Akt, Caspase-3 are either very dim or no signal at all. I increased loading from 30ul to 50ul and got the same disappointing results. I saw some references showing western using primary neurons and seems strong signals. Is there a tricky part about doing western using primary neurons? Anybody has experience on that? I appreciate your input very much!

I cultured primary neurons on 6-well plates and use 150ul lysis buffer per well (My guess is that neurons are much less dense than HEK293 cells so that the protein levels would be lower in lysate), western is done with Cell Signaling antibodies, Li-Cor 2nd antibodies and imaging system.

#2 GNANA

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Posted 20 February 2013 - 02:40 PM

your issue is mostly due to the very low quantity of protein used. 150 microlitre per well is too much, i would suggest not more than 50 micro litres/well, doing so you could able to load the maximum concentration obtained.
I would prefer being perfectionist rather than a passionist in Research.

I always had an alternate hypothesis....




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