Lab 02
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4 Lab 02: Measurement and the Standard Curve Experiment 1 Standard Curve Table 1 Tube Final Protein Concentration
µL of
BSA protein stock (0.5 mg/mL) needed
Bradford Reagent
0.15M NaCl for 1.0 mL final volume
8 50 µg/mL 100 uL 900 µL 0 uL 7 25 µg/mL 50 uL 900 µL 50 uL 6 15 µg/mL 30 uL 900 µL 70 uL 5 10 µg/mL 20 uL 900 µL 80 uL 4 7.5 µg/mL 15 uL 900 µL 85 uL 3 5.0 µg/mL 10 uL 900 µL 90 uL 2 2.5 µg/mL 5 uL 900 µL 95 uL 1 0 µg/mL 0 uL 900 µL 100 uL Table 2 Final Concentration
Absorbance 595nm
50 µg/mL 0.96 nm 25 µg/mL 0.76 nm 15 µg/mL 0.63 nm 10 µg/mL 0.28 nm 7.5 µg/mL 0.27 nm 5.0 µg/mL 0.16 nm 2.5 µg/mL 0.10 nm 0 µg/mL 0 nm Part B: Protein Concentration in Milk Table 3 My Milk Type = ____2%______ The Dilution of milk I used was: ___1:100________ Tube Unknown Volume Bradford Reagent 0.15M NaCl for 1.0 mL final A
595n
m Concentration Measured by Spec. Concentration of Unknown Volume 1 5 µL 900 µL 95 uL 0.06 1.527 ug/mL 30.540 ug/uL 2 10 µL 900 µL 90 uL 0.20 5.089 ug/mL 50.890 ug/uL 3 20 µL 900 µL 80 uL 0.23 5.852 ug/mL 29.260 ug/uL 4 40 µL 900 µL 60 uL 0.86 21.883 ug/mL 54.708 ug/uL a. What was your final R
2 value? What does this tell us about the precision of your measurements? The R^2 value that I got was 0.8656. After excluding the last 2 values the R^2 value changed to 0.9435. This calculation indicates how closely our data is to a fitted regression line. A R^2 value between 0.98 and 1 is considered ideal. This suggests that human errors such as pipetting techniques occurred during the lab and the absorbance measurements are not precise. Due to this, there is a possibility that the calculated unknown protein concentration will differ from the actual value.
5 b. Look at the final two values of your standard curve on the scatterplot, do they correlate strongly with the rest of your data? Why or why not? No, although they are on trend, they are not as linear as the other data points. This is because the Bradford reagent saturated, causing the trendline to level off. c. What happens to your R
2 values if you exclude the last two values in the curve? Does that change the concentrations of your unknown? If so, which should you use to calculate the unknown concentration in milk? The R^2 values increased closer to 1 (from 0.8656 to 0.9435). Consequently, by excluding the final two values, a different linear regression equation is generated with a different slope value. Thus, this changes the values for the concentration measured by the spectrum and thus the overall value for the concentrations of the unknown. The edited standard curve should be used over the original standard curve to calculate the unknown concentration of milk protein, because the R^2 value is higher. This suggests that the trendline better fits the first few datapoints and the regression is more precise. d. What is the concentration of the milk solutions in the cuvette? Show your work in what you hand in (it’s worth points). Either construct what you did digitally (2+2)/4=1, or take a photo of your work and insert it into the document. Original Standard Curve Concentration measured by spec. = abs/slope C= 0.06/0.0196 C= 3.06122449= 3.061 ug/mL Edited Standard Curve Concentration measured by spec. = abs/slope C= 0.06/0.0393 C= 1.526717557= 1.527 ug/mL e. What is the concentration of protein in your milk? Did you use all data in calculating the unknown concentration? If you excluded some data, provide a justification. Remember you must account for dilution. Show your work. Original Standard Curve c1= x v1= 5 uL c2= 3.061 ug/mL v2= 1 mL c1v1=c2v2 (x)(5 uL)= (3.061 ug/mL)(1mL) X= 0.6122 x 100
6 X=61.22 ug/uL Edited Standard Curve c1= x v1= 5 uL c2= 1.527 ug/mL v2= 1 mL c1v1=c2v2 (x)(5 uL)= (1.527 ug/mL)(1mL) X= 0.3054 x 100 X=30.54 ug/uL Average milk concentration using original standard curve= (61.22 ug/mL+ 102.14 ug/mL + 58.675 ug/mL + 109.695 ug/mL)/ 4 = 82.9325 ug/uL Average milk concentration using edited standard curve= (30.540 ug/mL+ 50.890 ug/mL + 29.260 ug/mL + 54.708 ug/mL)/ 4 = 41.3495 ug/uL The last two data points were excluded, as the R^2 value was closer to 1. This suggests that the trendline better fits the first few datapoints and the regression is more precise. This will give us a more accurate unknown concentration as seen above. Thus, the edited standard curve equation was used to calculate the concentration measured by spec and the concentration of unknown volume in Table 3. f. How does your determined concentration compare to the concentration of protein listed on the milk carton? The original concentration of protein listed on the milk carton was 33.3 ug/uL. The determined concentration was 41.3495 ug/uL. This is higher than the real concentration of protein, suggesting that the measurements used for the standard curve were not precise enough. Human error occurred during the lab such as not pipetting carefully, thus causing inaccurate absorbance values. g. Turn in this Data Sheet package (pages 5 and 6) along with a copy of your graphs that you plotted in Excel. Make sure that your graphs display the trend line, the equation for the line, and the R
2 values.
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7 y = 0.0196x + 0.1135
R² = 0.8656
0
0.2
0.4
0.6
0.8
1
1.2
0
10
20
30
40
50
60
Absorbance (nm) Final Concentration (ug/mL)
Standard Curve with Bradford Reagent y = 0.0393x - 0.0217
R² = 0.9435
-0.1
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0
2
4
6
8
10
12
14
16
Absorbance (nm)
Final Concentration (ug/mL)
Standard Curve with Bradford Reagent Edited
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