question about TNF alpha western blot

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beautyazhu
beautyazhu's picture
question about TNF alpha western blot

 I am looking for the mTNF-alpha in b16f10 cell lines transient transfected with pDNA, I collected the cell lysate and concentrated medium to run western blot.  The result is not good, the signal of the bands are not sharp and there were no difference between the control cells and transfected cells.....there are bands around 25kda and 50kda......
Do anyone has some suggestions? I will appreciate it a lot! thanks

samm
samm's picture
You may actually be seeing

You may actually be seeing TNFa! Its ~25-26 KDa initially, gets processed to the most active ~17 kDa form. It also has trimeric 51 kDa structures (which dissociate easily), and has a soluble receptor at ~55 kDa.
What Ab are you using?
And incidentally, is there a particular reason you are not using an ELISA - the sandwich ELISAs are pretty specific nd sensitive, and you can use ELAST to further imrove sensitivity, though I do not think there is any biological significance at those low amounts (essentially baseline for a given cell/tissue type, since many cells can hold preformed TNFa).
Best,
-sam

beautyazhu
beautyazhu's picture
Hi Sam,

Hi Sam,

Thank you for the reply!
We are using the purified Anti-mouse TNF-alpha antibody , is the TNF protein mainly exist in the cytosome or excrete to the medium? I can not see clear band either in the cell lysate or condensed medium.
http://www.biolegend.com/purified-anti-mouse-tnf-alpha-antibody-979.html

There is no particular reason that we do not use ELISA, just because WB is more available in our lab. I am not major in this, just for one time detection.

Thanks again for your help.

Yu