Good Evening,

I have a question about the normalisation of my results when measuring phoshorylated proteins.

Example:

EGF stimulation experiment:

I measure phoshorylation of AKT as a function of EGF stimulation time (0=control, 5min, 10min, 15min, 30min, 60min):

1. so I start with densitometric evaluation of pAKt and its unphosphorylated version (AKT).

2. then I deal with the charge control (VInculin) and divide each individual band of the charge control by the highest value of the charge control and thereby obtain my individual normalisation factors.

3. now I divide the single pAKT values by the single normalisation factors and divide the single AKT values by the single normalisation factors

4. finally, I divide the normalised pAKT values by the normalised AKT values.

5. i relate the values to my control normalised to =1 and get the relative change.

Now my questions:

- Isn't (pAKT/Vinculin) : (AKT/Vinculin) ultimately the same as pAKT/AKT? How can I still usefully normalise with my charge control like this?

- Assuming that the unphosphorylated form (AKT) is poorly or not at all densitometrically evaluable, may I replace AKT with my charge control in this "emergency" and divide pAKT by Vinculin?

Many thanks in advance!!!