1. Why do you recommend normalization to total protein mass per well rather than reporting interpolated concentrations?

Normalization to total protein mass per well allows you to compare values for samples where different amounts of total lysate protein have been added to the assay well.    

2. Can I normalize to cell number if I have not quantified the protein concentration after preparation of cell lysates?

We strongly recommend quantification of total protein mass. However, you may normalize values to estimated cell numbers by reporting Akt concentrations per number of cells. Due to potential variability of cell growth in tissue culture plates, inter-well measurement variability may be higher using this normalization method.

3. How should I calculate relative % phosphorylation if I run the same sample set in the singleplex Phospho Akt (Ser473) Kit and the singleplex Total Akt Kit?

After normalization to the well-specific protein concentration, divide the units of phospho-Akt (U_P473) by the pg total Akt. This will provide a measurement of units of phosphorylation per pg Akt.

[(U_P473/µg protein)/(pg total Akt/ µg protein) * 100] = U_P473/pg Akt