Bradford Assay-Standard Curve 0.800 0.700 0.600 0.500 0.400 0.300 y = 0.0148x - 0.003 R? = 0.9981 0.200 0.100 0.000 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55 00 -0.100 Protein amount in µg Absorbance
The purification of cytochrome C begins with 1) yeast homogenization using a bead beater in the presence of BME (a reducing agent) and a protease inhibitor from approximately 900 grams of Baker’s yeast. Then, 2) insoluble cell contents were removed by centrifugation at 4,000 x g for approximately ten minutes. The ruptured cells (lysate) after centrifugation had a total volume of 0 mL and a 1.0 mL aliquot was set aside for further analysis. The following data was obtained from the 1.0 mL aliquot to quantify the protein amount and purity:
- The absorbance at 410 nm of the aliquot was 0.460 (1 cm pathlength).
- The absorbance at 595 nm from a 1.0 mL Bradford Assay solution that was diluted by 250-fold from the aliquot was 0.681 (1 cm pathlength).
Using the information given,
- Calculate the total protein amount in mg from the absorbance at 595 nm.
- Calculate the cytochrome C amount in mg from the absorbance at 410 nm using Beer’s Law.
![Bradford Assay-Standard Curve
0.800
0.700
0.600
0.500
0.400
0.300
y = 0.0148x - 0.003
R? = 0.9981
0.200
0.100
0.000
0.00
5.00
10.00
15.00
20.00
25.00
30.00
35.00
40.00
45.00
50.00
55 00
-0.100
Protein amount in µg
Absorbance](/v2/_next/image?url=https%3A%2F%2Fcontent.bartleby.com%2Fqna-images%2Fquestion%2F9b60de5f-8fb0-4e3b-8258-2de6c1a93a8c%2F45b1fcee-9358-4126-8717-997a3c061927%2Fkefzxz_processed.png&w=3840&q=75)
![Protein amount in mg
Protein amount in ug
Absorbance
0.0000
0.00
0.000
0.0025
2.50
0.031
0.0050
5.00
0.075
0.0100
10.00
0.134
0.0150
15.00
0.229
0.0200
20.00
0.280
0.0250
25.00
0.386
0.0375
37.50
0.540
0.0500
50.00
0.742](/v2/_next/image?url=https%3A%2F%2Fcontent.bartleby.com%2Fqna-images%2Fquestion%2F9b60de5f-8fb0-4e3b-8258-2de6c1a93a8c%2F45b1fcee-9358-4126-8717-997a3c061927%2F9d6flyf_processed.png&w=3840&q=75)
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Spectrophotometric analysis can be carried out to find the concentration (or amount) of protein in a sample. To find the concentration (or amount) of protein in our sample, first we need to draw a standard curve using known concentrations of protein and the absorbance values at those concentrations. Then we generate a trendline (the dotted blue line in the Bradford assay-standard curve) and generate the equation the line. The general equation of a line is ' y= mx + c ' .
Here;
'y' is the Y-axis value ( Absorbance at 595nm in the Bradford assay-standard curve)
'x' is the X-axis value ( amount of protein in in the Bradford assay-standard curve)
'm' is the slope
'c' is the Y-intercept.
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