Nuclear extract loading control in Western blot - (Jul/18/2008 )
I am in front of a theoretical question. I would like to use a loading control for nuclear extract.
I asked in different lab and nobody has a perfect answer.
lamin: but is essentially on the nuclear membrane. I am not sure that I will have it in my lysat since I centrifuge the nuclear membrane and take the supernatant.
Histone H1: is the antibody working well?
TBP: do anybody has experience with it?
beta-tubuline: in my case is not working and I have strong doubt that if I see something this is because of cytoplasmic contamination.
PCNA: not working in my cells, and furthermore is dependent of proliferation so that it is a bad control for antiproliferation treatment of cells.
So do anybody of you have a good advice, if yes can you please tell me the antibody you use (company)and the concentration you dilute.
Thanks a lot
I've used abcam TBP antibody (ab818) before. In my experience, a dilution of 1:200 in TBST-1%BSA applied overnight at 4C worked reasonably well. That means I got strong bands somewhat below 37kDa after 1 min exposure on high-sensitive Amersham film and Amersham ECL. There was a weak band below the specific one, but no others and little background.
The bands were quite equal in different lanes, it appeared to be useful for loading control in my experiments.
However, the cytoplasmic fraction had TBP signal as well, although to a much lesser extent.
I didn't find it a perfect ab, but maybe worth trying it (after you checked the literature there is no change expected in your conditions).
I have successfully used p84(abcam) as nuclear loading control in primary B-cells. i had not detected it in my cytoplasmic extract.
Thanks for your answers