I am trying to verify if ghrelin is present in cortical collecting duct cell lines. Ghrelin is approximately 3 kDa and difficult to find on western blot. Does anyone have any suggestions? Thanks!
Two possible approaches: (1) Use a Tris-tricine gel, which is optimized for small peptides, and a short transfer time (2) use a sandwich ELISA instead, if the antibodies are available.
I have successfuly used self cast bis-tris gradient (15-20%) gels (Nu-PAGE) for the separation of trypsin digest.
http://openwetware.org/wiki/Sauer:bis-Tris_SDS-PAGE,_the_very_best
Hi,
I had problems with a 4 kDa protein using conventional method for western blot, but then I tried Tricine-SDS-PAGE (see file attached) and it worked very well.
Hope this can be useful.
Ana
thank you all for all of your answers. I will look at each and try your suggested protocols. Thank you!
I agree to the others, try Bis-Tris or Tris-Tricin gels. If possible I suggest you to buy them first (or ask for one in another institute/department :-)) to eliminate problems due to self-cast gels. If it works, you can think about protocols for making them yourself to save money.
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