KABIGTING, Joseph John Patrick D.O.

O3A – B.S. Medical Technology

Sir Francisco Elegado and Ma’am Eureka Teresa Ocampo
May 4, 7 2022
MOLECULAR BIOLOGY AND DIAGNOSTICS
LAB EXERCISE ON USE OF STANDARD CURVE FOR PROTEIN DETERMINATION

OBJECTIVES
This exercise will allow the student to:
1. Prepare a protein standard curve given the absorbance of the protein standards
2. Determine protein content of a given sample using the standard curve data
3. Analyze results
OUTPUTS:
1. Filled up Tables 1 and 2
2. Line graph with regression equation
3. Calculated protein concentrations of samples 1-5 in Table 2
Methodology
1. Study Table 1 which presents the absorbance reading of 3 replicates of bovine serum
albumin or BSA, the most common protein standard used for protein concentration
determination
2. Take the average absorbance values of the 3 replicates
3. Using MS Excel, chart the average absorbance value in the Y axis and the BSA concentration
on the X-axis. Use scatter graph
4. Then click on inside the chart area, and select add trendline (linear)
5. Set intercept, display equation and R 2 value on chart. Take a screenshot of the graph and
paste on the file that you will submit as part of your assignment.
6. The equation is y = mx t b, where m is the slope and b is the y-intercept and the value of the
regression coefficient R2. You will notice that since the graph is highly linear, there will be no
y-intercept
7. Determine the protein content of the samples in Table 2 using the equation of the standard
curve obtained from data in Table 1.

Table 1. Absorbance readings of BSA at different concentrations.

Absorbance at 595 nm
BSA concentration (μg/mL)
Average
Rep 1 Rep 2 Rep 3
Absorbance
0 0.000 0.000 0.000 0.000
5 0.191 0.184 0.215 0.197
10 0.340 0.345 0.369 0.351
15 0.536 0.575 0.553 0.555
20 0.694 0.701 0.705 0.700
25 0.809 0.811 0.844 0.821
30 0.971 0.973 1.032 0.992
Screenshot:

Standard Curve
1.2

1
f(x) = 0.0338923076923077 x
R² = 0.998138519947514
0.8

0.6

0.4

0.2

0
0 5 10 15 20 25 30 35

Table 2. Absorbance readings of samples with unknown protein concentrations

Sample # Absorbance 595 Calculated Protein

Trial 1 Trial 2 Trial 3 Average content (μg/mL)
1 0.548 0.54 0.544 0.544 16.047
2 0.36 0.36 0.366 0.362 10.679
3 0.61 0.612 0.614 0.612 18.053
4 0.25 0.248 0.25 0.249 7.345
5 0.789 0.79 0.79 0.790 23.304