Question

A protein assay requires the reaction of the protein solution with a protein-binding dye for one minute, followed by measuring the absorbance of the protein-dye complex at 505 nm. The absorbance data for four standard protein solutions and the blank is provided in the table below. The absorbance of each solution was measured three times. Construct a calibration curve using the data in the table, and find the slope (m), intercept (b), and their respective standard deviations using the method of least squares. Note that the calibration curve will be created from absorbance values corrected for the absorbance of the blank and not the raw data in the table (including individual blank values). When creating the calibration curve, do not use the average absorbance at each protein concentration. Instead, use each data point separately, meaning there will be three data points at each protein concentration, for a total of 15 points (including each individual blank value).

	replicate 1	replicate 2	replicate 3
blank	0.0332	0.0331	0.0335
0.10 g/dL	0.1002	0.0999	0.998
0.20 g/dL	0.233	0.233	0.233
0.50 g/dL	0.532	0.534	0.533
1.00 g/dL	1.032	1.033	1.031

m= 1.012 +/- ?

b= ? +/- ?

Calculate the concentration and uncertainity of a protein solution that produced an averge absorption of 0.470 when measured three times.

number = ? +/- ?

Answer 1

Answer:

The following procedure can be used for constructing a calibration curve:

Step 1 Prepare known samples of analyte covering a range of concentrations expected for unknowns. Measure the response of the analytical procedure to these standards to generate data like the left half of Table-1.

Step 2 Subtract the average absorbance (0.0993) of the blank* samples from each measured absorbance to obtain corrected absorbance. The blank measures the response of the procedure when no protein is present.

Step 3 Make a graph of corrected absorbance versus quantity of protein analyzed (Figure-2). Use the least-squares procedure to find the best straight line through the linear portion of the data, up to and including 20.0 microgram of protein (14 points, including the 3 corrected blanks, in the shaded portion of Table-1). Find the slope and intercept and uncertainties with Equations A, B, C D, E & F. The results arem = 0.016 30 sm = 0.000 22 sy = 0.0059 b = 0.0047 sb = 0.0026 The equation of the linear calibration line is