Which control protein for Western Blotting of nuclear protein? - (Jul/09/2008 )

Hi all,

I'm running Westerns on nuclear proteins. Would like to find out which housekeeping proteins do you guys use to ensure equal protein loading?

I've tried histone, but it does not work well.

Is there beta-tubulin in the nucleus?

thanks!

regards,
shyn

You may have problems with PCNA since its expression is cell cycle regulated and is not a housekeeping gene. PCNA is also quickly degraded when DNA damage pathways are activated. If your experiments have any cell cycle affects or DNA damage this will not work as an appropriate loading control. Rather, I would recommend using lamin A/C.

I agree with cellcounter. PCNA. It is abundant and there are good Abs available. And it does not vary with the cell cycle (in mammalian cells), but it is more abundant in proliferating cells compared to quiescent.

rkay is also right that it may be better to find a structural protein to use as a control. But westerns aren't really that quantitative anyway, so just pick something that works. Even staining the blot afterwards works.
-d

Nucleolin - nuclear only, sutructural protein.

thanks all for your helpful suggestions.

I'll try beta-actin as we have it in the lab.

Has anyone tried beta-tubulin? or alpha-tubulin? Or can anyone advise me if tubulin is present and stable in the nucleus?

thanks alot!

shyn

We usually use beta tubulin to check the purity of our nuclear fractions. If tubulin shows up, your fractions are no good. I use PCNA and H3 as nuclear loading controls.

You can use Topoisomerase I and TATA binding proteins as loading controls for nuclear proteins. Beta actin is not an ideal loading control for nuclear proteins. I hope this helps.
Tulip

P84 is widely used as a nuclear marker