PROBLEM: Western blot of a 220 kd nuclear protein - (Jun/13/2005 )
We tried several times to prepare a western blot of a 220 kd nuclear, low abundance protein (transcriptional factor) exprimized in a mouse cell line.
Surprisingly we just get a band in the range of 20 kd - our protein is missing !!!!
We have also a high background.
Does anybody have an idee ?
1. Protein isolation: RIPA buffer
2. PAGE: 6% running gel, 4,5% stacking gel
3. Wet blotting buffer: glycin, Tris, EDTA, no SDS
blotting over night, 12 V, 14h,
4. blocking: 5% non fat milk
HRP; biotin-avidin labeling for the marker
6. Substrate: PIERCE chemoluminesce
Do you have a positive control?
My experience is that it is a good idea to enrich for nuclear proteins when
probing for low abundance nuclear proteins. For example, I have previously found that
35-50 micrograms of nuclear extract will give me a band of equal intensity as using 150-200 micrograms of whole cell extract.
As for the background problem, first detect the protein, then clean it up.
I'm sure there are an infinite number of posts on this site about effieciently cleaning up Western blots.
i've noticed that some antibodies works better in PBS rather than in TBS... But i agree with mikew. You should use extraction of separate nuclear and cytoplasmic proteins.
Thank u both very much !
We do not use a positive control till now.
My thought was also that probably there is a problem with the protein isolation, because we overexprimized the protein, but maybe the amount is still low.
But probably the RIPA-buffer is also not the best solution for nuclear proteins.
Do u have a protocol for nuclear extract preparation or a good protocol for cell extracts?