Loading controls for neuritogenesis

[AvengingAnjel]

Hi,

I am looking at neurite outgrowth for PC12 and SHSY5Y cells.
I have 2 questions:

1. I am interested in the phosphorylation status of certain genes such as Erk and Creb. Can someone clarify if using the non-phosphorylated proteins as loading controls is sufficient, even at later time points after treatment (24h-72h)?

2. If I want to measure expression of neuronal markers such as beta3 tubulin and GAP43, what loading controls are good? Are there any publications supporting this?

Thanks!

[Drekketh]

Hey AA,
as it comes with every phosporylation study you require at least two antibodies for your Western Blot.
First one is an antibody against your phosphorylated protein of interest and the second one against the total protein. So in that case u need anti-phospho Erk and anti-phospho CREB and antibodies against the total Erk and CREB.
Loading simply "unphosphorylated" protein sample and screening with a phospho Ab does not control properly the phorphorylation status of your gene. You need to have the total Ab also.

[AvengingAnjel]

Hi Drekketh,

Thank you for your reply
Let me rephrase this to make sure I got it right.
To look at phosphorylation status of Erk, I need:
1. phospho-Erk antibody
2. Total Erk antibody
Do I also need a normal housekeeping gene like GAPDH or actin (especially for time points past 24h)?

Is there a good housekeeping gene I could use for measuring expression of other neurite specific factors such as GAP43 and beta tubulin?

THANKS!!
AA