# bicinchoninic acid (BCA) protein assay

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Blood
Trace
Negative
Trace
Negative
Negative
The bicinchoninic acid (BCA) protein assay enables the quantification of protein by measuring the absorbance of a BCA : protein
complex at 562nm. Below is a table containing absorbance readings at 562 nm for standard protein samples of known protein
concentrations.
Table 2: Absorbance readings at 562 nm for six standard and two test samples
Sample Tube: Absorbance Absorbance Absorbance Protein
ml
Standard 1:
1.141
1.253
1.080
1000
Standard 2:
0.946
0.806
0.822
500
0.393
Standard 3:
Standard 4:
0.390
0.270
250
125
0.296
Standard 5:
0.166
0.144
25
0.453
0.300
0.161
0.128
1.078
0.288
Standard 6:
0.136
0.120

Test Sample 1:
Test Sample 2:
1.141
0.296
1.252
0.270
Unknown
Unknown
a) Calculate the mean absorbance reading for each sample (standards 1-6 and test samples 1 and 2). Show all your working and
present your data in table format.
b) Calculate the normalized absorbance reading for each standard sample by subtracting the mean value for sample 6 from each
sample’s mean value. Show all your working and present your data in table format.
c) Why is it necessary to subtract the mean value for standard 6 from each sample’s mean absorbance reading?
d) What happens to the absorbance values at 562 nm as the protein concentration of the standard samples increases?
e) Why are you measuring the absorbance of these samples at 562 nm and not 280 nm? Reference your answer
Plot a graph where the x-axis represents Protein Concentration (ng/ml) and the y-axis represents Normalized Absorbance values at
562nm. Work out the equation for the line of best fit.
Using the equation of the line of best fit, calculate the concentrations of proteins in test samples 1 and 2. Show all your working.
The enzyme salivary amylase is responsible for the breakdown of starch to maltose in the mouth. Experimentally the presence of starch is
determined using the Lugol’s test and the appearance of maltose is determined by the Benedict’s test. Four solutions were tested for the
presence of starch and maltose using these tests. The results are listed in table 3.
For the Benedict’s test a blue colour indicated the absence of maltose
a green colour indicates the presence of a tiny amount of maltose
a yellow colour indicates the presence of some maltose
a red colour indicates the presence of a lot of maltose
For the Lugal’s test a purple – black colour indicates the presence of a lot of starch
a purple – blue colour indicates the presence of a small amount of starch
a yellow – orange indicates the absence of starch
Using this information deduce which tube(s) contained the most starch following incubation?
Which tubels) contained the most maltose?

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