Question

You are performing a Bradford assay. You construct your standard curve (concentration in mg/mL on the x-axis, Absorbance on the y-axis) and derive a trendline with an equation of y = 0.25x (the intercept is set to zero because you blanked your spectrophotometer correctly). You dilute protein A using the volumes given below, to a final volume of 1000 μL, and measure the absorbance:

6 μL to 1000 μL = Abs 0.04

80 μL to 1000 μL = Abs 0.15

600 μL to 1000 μL = Abs 0.95

What is the concentration of protein A in mg/mL?(Assume a 1 cm path length).

Answer 1

According to Beer-Lambert’s law, Absorbance is directly proportional to the concentration of solution at constant path length.

A directly proportional to C (concentration)

A = e x C

Now the equation of a graph line is y = 0.25 x X i.e a straight line passing through origin( y = mx) hence slope m = 0.25 gives molar absorptivity (e). as its for plot of Absorbance (on Y-axis) verses concentration (on X-axis).

Now we have A = 0.04 for C=? e = 0.25

A = e x C

C = A / e

    = 0.04 / 0.25

    =0.16 mg /microL

    =0.16 mg x 10³ /mL………..(1 microL = 10^-6L= 10^-3 mL) (reciprocated)

C =160 g/mL