WB for secreted protein? - (May/30/2007 )
Hi,
I have limited experience with Western blotting, but all of the proteins that I've looked at are either nuclear or cytoplasmic. Now I need to look at a cytokine, that is secreted (my samples are cells in cell culture). I was wondering:
a] can I just do a western blot on whole cell extract for this protein - is this generally acceptable to reviewers?
b] Can I just take culture medium and load it onto my gel and blot, or do I need to concentrate first? I'm guessing that ELISA may be a better idea? But this seems much more expensive and I only have a couple of samples...
Thanks!
this may be helpful:
http://www.protocol-online.org/forums/inde...=22235&st=0
I have limited experience with Western blotting, but all of the proteins that I've looked at are either nuclear or cytoplasmic. Now I need to look at a cytokine, that is secreted (my samples are cells in cell culture). I was wondering:
a] can I just do a western blot on whole cell extract for this protein - is this generally acceptable to reviewers?
b] Can I just take culture medium and load it onto my gel and blot, or do I need to concentrate first? I'm guessing that ELISA may be a better idea? But this seems much more expensive and I only have a couple of samples...
Thanks!
ELISA should be more sensitive and better to analyze many samples than Wb; if you down-scale to 384-well ELISA plates you will save material
I have limited experience with Western blotting, but all of the proteins that I've looked at are either nuclear or cytoplasmic. Now I need to look at a cytokine, that is secreted (my samples are cells in cell culture). I was wondering:
a] can I just do a western blot on whole cell extract for this protein - is this generally acceptable to reviewers?
b] Can I just take culture medium and load it onto my gel and blot, or do I need to concentrate first? I'm guessing that ELISA may be a better idea? But this seems much more expensive and I only have a couple of samples...
Thanks!
If you only want to look your protein secretion ( without quantative determination of your protein) in media it will be good enough to do WB ( 7-12 samples you can do with one PAAG) If your cytokine secreted in very low concentration may be it will be reasonable to concentrate your media sample ( for ex 1 ml) on microcon with suitable Mw cutoff before WB.
Funny thing! I tried a WB on culture media this week for adiponectin, a cytokine secreted from adipocytes. All I did was to determine the protein concentration of the media by standard Bradford and blotted 50 ug of total protein. Worked just fine!
Thanks for all of your replies, I think that I will try a straight western first, of both cells and medium, if that doesn't work then look into the ELISA.