Quantitative Determination of Protein concentration using the biuret test
Winny Stephanie
Experiment 1: Quantitative Determination of Protein Concentration Using the Biuret Test
Table 1: Experimental protocol for construction of the protein standard curve
Tube
1
2
3
4
5
6
Buffer (ml)
1.0
0.8
0.6
0.4
0.2
0.0
BSA Protein solution (10 mg ml-1) (ml)
0.0
0.2
0.4
0.6
0.8
1.0
Biuret reagent (ml)
4.0
4.0
4.0
4.0
4.0
4.0
Total Volume (ml)
5.0
5.0
5.0
5.0
5.0
5.0
Final protein concentration (mg ml-1)
0
2
4
6
8
10
Absorbance
0.000
0.092
0.163
0.272
0.363
0.474
Table 2: Experimental protocol for determining the protein concentrations of silverbeet grown in the sun and shade
Sun
Shade
Column for modified procedure(s)
Procedure
Protein concentration (ml)
1
1
0.4
0.4
Biuret reagent (ml)
4
4
4
4
Absorbance
1.104
0.908
0.465
0.363
Protein concentration derived from standard curve (mg ml-1)
-
-
10.2
8
Protein concentration of original, unmodified solution (mg ml-1)
25.5
20
-
-
Figure 1. Determination of Protein Concentration of Solution in Standard Curve
Q4. The graph should be based on the line of best fit, as the straight line is one of a method to smooth out the uncertainties or the experimental errors and it will give ab accurate reference to determine the unknown protein concentration of the unknown samples. However, if the line connect to all the data points, the trend in between will be a straight line, which is not justified.
Q5. It is not possible to determine the protein concentration if the measured absorbance is off scale. Because the standard curve constructed has a specific range, therefore the concentration cannot be determined when the absorbance is out of the range. Even if it could be calculated, the concentration determined will not be accurate.
Q6.
a. I would decrease the amount of the protein solution to 0.4 ml and dilute it with 0.6 ml of buffer solution, however the volume of reagent will remain 4.0 ml, and the total of solution would be 5.0 ml. then, the unknown protein concentration can be determine by
Experiment 1: Quantitative Determination of Protein Concentration Using the Biuret Test
Table 1: Experimental protocol for construction of the protein standard curve
Tube
1
2
3
4
5
6
Buffer (ml)
1.0
0.8
0.6
0.4
0.2
0.0
BSA Protein solution (10 mg ml-1) (ml)
0.0
0.2
0.4
0.6
0.8
1.0
Biuret reagent (ml)
4.0
4.0
4.0
4.0
4.0
4.0
Total Volume (ml)
5.0
5.0
5.0
5.0
5.0
5.0
Final protein concentration (mg ml-1)
0
2
4
6
8
10
Absorbance
0.000
0.092
0.163
0.272
0.363
0.474
Table 2: Experimental protocol for determining the protein concentrations of silverbeet grown in the sun and shade
Sun
Shade
Column for modified procedure(s)
Procedure
Protein concentration (ml)
1
1
0.4
0.4
Biuret reagent (ml)
4
4
4
4
Absorbance
1.104
0.908
0.465
0.363
Protein concentration derived from standard curve (mg ml-1)
-
-
10.2
8
Protein concentration of original, unmodified solution (mg ml-1)
25.5
20
-
-
Figure 1. Determination of Protein Concentration of Solution in Standard Curve
Q4. The graph should be based on the line of best fit, as the straight line is one of a method to smooth out the uncertainties or the experimental errors and it will give ab accurate reference to determine the unknown protein concentration of the unknown samples. However, if the line connect to all the data points, the trend in between will be a straight line, which is not justified.
Q5. It is not possible to determine the protein concentration if the measured absorbance is off scale. Because the standard curve constructed has a specific range, therefore the concentration cannot be determined when the absorbance is out of the range. Even if it could be calculated, the concentration determined will not be accurate.
Q6.
a. I would decrease the amount of the protein solution to 0.4 ml and dilute it with 0.6 ml of buffer solution, however the volume of reagent will remain 4.0 ml, and the total of solution would be 5.0 ml. then, the unknown protein concentration can be determine by