Concentration UV vs Amino acid analysis

Hi Arnoud,
I have a question in regards to concentration. I originally received 100 ug/mL as the concentration of the Analyte (Ag) performed by UV spec. And then proceeded making dilutions of my Analyte using this value. Then I got a result by amino acid analysis of 138 ug/mL a week later. I already performed my assay using the UV spec result of 100ug/mL with I performed my dilutions. I re-processed the data using the AAA concentration and noticed a difference in the ka (8.84E5 using the UV conc.) vs ka (1.99E6 using the AAA concentration). For KD's, using the UV conc. value was 1.09E-10, compared to a KD of 8.27E-11 using the AAA values. I suppose my question is, would I trust the UV or the AAA concentration? kd was essentially the same for both. Thanks.

Hi,
Analyte quantification is always surrounded with some variation. When you measure the concentration based on the UV (A280) you should use the molar extinction coefficient of that analyte to calculate the concentration. For an antibody this is around 1.4 (E 1 mg/ml / A280 nm) but variations occur.
The amino acid analysis depends on a calibration curve and possibly has its own region of variation.
I can imagine that when you do a Bradford or BCA protein concentration assay that you also will find slight variations in concentration depending on the calibration you use.
Bottom line is that a thorough exact concentration determination is very difficult.

There is a PDF document about this on the SPR-pages
www.sprpages.nl/downloads/how-to#Tprotein

As for your results I would say that that the k_a is around 1E6 M^-1s^-1 (2 significant digits is too much) and the K_D around a 100 pM.

Kind regards Arnoud