Western Detection of Transcription Factors - -more difficult than other proteins? (Sep/29/2006 )

Hi everybody,

I have been trying to detect my protein of interest by western blotting in the course of expression analysis in human melanoma cell lines. RNA levels are very high, so protein levels might be as well ( i know that this doesnt have to be necessarily the case). My protein is a transcription factor.

anyway, i have tried 4 different commercial antibodies and various methods of blocking for months now, and either i dont get any signal or tons of unspecific stuff.

at a recent meeting, some guy told me that the problem might be that transcription factors by nature tend to relocate to the nucleus and might stick around there. I already had tried nuclear extracts before and did not get any result.

are TFs more tricky? Is there a certain trick to them? How about repeated denaturing of the samples?
does anybody have experience with that?

Thank you so much!!!

Tobias

Lyse your cells, spin down your extract for 10 min at max rcf at 4oC. Recover supernatant and pellet. Resuspend pellet in 10ul of 8M urea+Laemmli and load lysate and pellet onto your gel.
Your TF could be in the unsoluble fraction (pellet). If this is the case, try sonication.
It is not a matter of TF, I work with TFs and I don't have this problem very often...

Hi,
in such a case i would suggest you to use Lumigen(amersham), a higly sensitive ECL which is designed to detect low amount of proteins on the membrane. IT REALLY WORKS . but take care to use very less dilutions of primary and secondary antibody for incubation to avoid high background.
ope it helps
Rajgene