[Chlamydomonas] Dot Blots

Sara Cline via chlamy%40net.bio.net (by cline.228 from osu.edu)
Thu May 5 18:31:52 EST 2011


  I was wondering if anyone had ever performed a Dot Blot of Chlamydomonas
whole cell extract directly on PVDF or nitrocellulose membrane.  I have a
175 kDa protein and I am not seeing anything when I do a western transferred
from a polyacrylimide gel.  My concern about the dot blot on whole cell
extract is the pseudo-peroxidase activity of unbound heme.  (My protein has
a very low expression and I am using an HRP conjugated secondary antibody.)
 Does anyone know if there is perhaps something like a chelator that I could
add to the protein to precipitate out the heme?  (I am going to try a pull
down, but if possible, I would like to see my protein without this step for
future experiments.)

Thank you in advance!
- Sara

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