What do the amino acids that react with the Bradford reagent and give a result for A280 have in common? Do you think all proteins have the same or different percentages of these reactive amino acids?  How might this affect the concentration determining assays?

Protein Quantification

Lab 3: Protein Quantification Report

Data you will need:

hen egg white lysozyme extinction coefficient: 36,000 M^-1cm^-1

hen egg white lysozyme molecular weight: 14,313 g/mol concentration of your lysozyme stock solution: mg/ml (get this from your TAs)

1. Based upon the A₂₈₀ value of your unknown lysozyme sample, and the Beer-Lambert law, calculate the lysozyme concentration of your unknown. Show your work below, and report the concentration in mM.
2. Fill out this table for each of your lysozyme stock solution dilutions:

3. Using EXCEL (or some similar program of your choice), plot A₅₉₅ vs concentration (in mM) for the samples in the above table. This is your lysozyme standard curve. Print out the standard curve and attach it below. Indicate on the graph the A₅₉₅ value of your unknown, and draw a horizontal line to the standard curve, then a vertical line from the curve down to the x-axis to read your unknown lysozyme concentration. Your standard curve must include x and y labels, and appropriate units! Record your unknown concentration here  (mM).
4. You have calculated the lysozyme concentration of your unknown solution by two different UV/VIS methods. Do they agree with each other? How much are they different from each other? Which method do you think is more accurate?
5. What do the amino acids that react with the Bradford reagent and give a result for A₂₈₀ have in common? Do you think all proteins have the same or different percentages of these reactive amino acids?  How might this affect the concentration determining assays?