western blot for nuclear and cytoplasm seperation sample - (Jul/24/2007 )

Hi, there,

I seperated the nuclear and cytoplasm proteins by home-made low-salt buffer and high-salt buffer. Western blot was done to detect one transcription factor. Unfortunately I never got any bands of the transcription factor, but the actin worked well. I wondered whether the low- or high-salt buffer interfered with western blot. Does anybody have any idea about it? Thanks a lot.


high-salt buffer:
HEPES 50 mM
KCl 400 mM
EDTA 1mM
EGTA 1mM
DTT 1mM
PMSF 0.5mM
Aprotinin 10 μg/ml
NaF 20 mM l
Na4P2O7 1mM
Na3VO4 1 mM
Glycerol 20 %

hi,
there may be several reasons for not detecting the transcription factors in the westerns:
1. the concentration of ur transcription factor may be very low
2. may be the nuclear proteins havent been eluted, make sure ur pellet is sticky after adding the high salt buffer.
3. make sure u give exactly 10s vortex after adding the detergent
and i personally dont use the sodium salts u using but still get good extracts for transcription factor.

When I did western blot for this sample, I found multiple bands just under the loading well. I am wondering that the sample didn't run on the gel. At the same time I run another samples but they worked well. so I don't think there is any problem with my running buffer or something technique problem.

hi
actin hows a very strong signal. Typicaldilution for TF is 1/2000 for start