Hi Xianqing,

Since your intention is to see the nuclear accumulation of your proteins of interest, you will need to make nuclear –cytoplasmic fractions from your cells and to study the expression levels of the target proteins via Western blot (WB). Alternatively, you can also study the expression levels through Immunocytochemistry/Immunofluorescence (ICC-IF) staining, but WB would be better if you want to go for quantification of the accumulated protein.

Your experimental design is great however, I would suggest you to go for different nuclear and cytoplasmic loading controls. GAPDH & HDAC1 both are the target genes of hypoxia – so I would not recommend you to use these as loading controls. Glucose 6 Phosphate Dehydrogenase (G6PD) is good for cytosolic fraction while Lamin B1, Histone H3 and Histone H1 are the most commonly used loading controls for nuclear fractions when working in experiments involving hypoxia.

Using densitometry, you should be able to normalize the expression values of your proteins of interest in test samples to of that of the control samples using the mentioned cytoplasmic & nuclear loading controls.

Novus team has compiled a list of over 20 different Frequently Asked Questions & Answers on Hypoxia& HIFs http://www.novusbio.com/support/hypoxia-and-hif-faqs

I am sure that you will find this information helpful and let me know if you need any other help in this regard.

Best Wishes

Subhash