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 10^{3} /mL………..(1 microL =
10^{-6}L= 10^{-3} mL) (reciprocated)

C =160 g/mL

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Absorbance Values for BSA Standard Curve
BSA Concentration
(mg/mL)
Absorbance Trial 1
Absorbance Trial 2
Absorbance Trial 3
Absorbance Trial 4
Absorbance Trial 5
Absorbance Trial 6
0.2
0.239
0.247
0.195
0.234
0.287
0.280
0.4
0.510
0.499
0.513
0.522
0.586
0.549
0.6
0.796
0.771
0.695
0.754
0.799
0.800
0.8
1.045
1.002
0.999
1.023
1.015
1.018
1.0
1.260
1.197
1.045
1.200
1.243
1.107
Absorbance Values for IgG Standard Curve
IgG Concentration
(mg/mL)
Absorbance Trial 1
Absorbance Trial 2
Absorbance Trial 3...

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