I am working with PC12 cells and would like to know what are the best conditions to perform western blot of alpha synuclein (14 kDa). Especially, regarding: best buffer for protein extraction: Ripa or Laemmli? Boiling or not boiling of the samples? Which membrane is more suitable: Nitrocellulose or PVDF? Thanks!
Hi Luciana!
I found several papers were they do Western Bloting of alpha-Syn. They don't use precisely PC12 cells but it might help you anyway.
In this one they boil the samples (in Laemli loading buffer), then use a 12% gel and transfer to a nitrocelulose membrane:
http://www.scigine.com/method.php?ID=243896
In this one they also use Laemmli loading buffer, but use a 10-20% gradient gel, and a PVDF membrane:
http://www.scigine.com/method.php?ID=346797
Finally in this one they also use a 12% gel, they use a buffer with SDS as well (I assume they use Laemmli buffer as well) and nitrocelulose membrane:
http://www.scigine.com/method.php?ID=545489
Personal note: unless you want to do an IP or something like that, I would go straight for the Laemmli, since you'll have to use SDS sooner or later (for SDS-PAGE).
Hope that helps, at least a little!
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