One quantitative method for the measurement of protein uses a reagent called Coo
ID: 52416 • Letter: O
Question
One quantitative method for the measurement of protein uses a reagent called Coomassie Brilliant Blue, which changes from a brown to blue color upon binding protein. The blue color can be measured using a spectrophotometer (see lab 6). A cell biology researcher wishes to know the concentration of protein in an unknown sample and performs the following experiment:
Protein Sample
Volume protein added
Volume water added
Absorbance @595 nm
500 ug/ml, protein standard
5 uL
995 uL
0.082
500
ug/ml,
protein standard
10 uL
990 uL
0. 166
500 ug/ml, protein standard
20 uL
980 uL
0.314
500 ug/ml, protein standard
50 uL
950 uL
0.814
500 ug/mL
protein standard
100 uL
900 uL
1.622
Unknown protein sample
10 uL
990 uL
0. 132
Unknown protein sample
100 uL
900 uL
1.362
Use this data to estimate the concentration of protein in the unknown sample as follows:
a. Calculate the amount of protein added in each case for the 500 ug/mL standard samples.
b. Calculate the concentration in mg/mL for each of these standard samples (hint:
use C1V1 = C2V2 to calculate the concentration of each standard after the water is added)
c. Construct a graph of Absorbance vs. concentration for the standard samples (only use the standards here, not your unknowns)
d. Use the slope of this graph to "back-calculate" the concentration of the unknown protein sample in dilution
e. Calculate the concentration of the original protein sample (undiluted)
## Example calculation for above:
a. amount of protein added = volume protein added x concentration added
= 5 uL x 500ug/mL = 5uL x 500ug/ 1000uL = 2.5ug
b. concentration of standard sample = amount protein added / volume of sample
= 2.5ug / (5uL + 995uL) = 2.5ug/ I000uL = 2.5ug/mL
c. see excel section in back of lab manual
d. equation of line: Abs = slope x [protein], so [protein] = Abs/slope = 0.082 / slope
e. original concentration = [protein] from (d) x 1000/5 (I000uL in dultion / 5uL of stock)
Protein Sample
Volume protein added
Volume water added
Absorbance @595 nm
500 ug/ml, protein standard
5 uL
995 uL
0.082
500
ug/ml,
protein standard
10 uL
990 uL
0. 166
500 ug/ml, protein standard
20 uL
980 uL
0.314
500 ug/ml, protein standard
50 uL
950 uL
0.814
500 ug/mL
protein standard
100 uL
900 uL
1.622
Unknown protein sample
10 uL
990 uL
0. 132
Unknown protein sample
100 uL
900 uL
1.362
Explanation / Answer
a. amount of protein added = volume protein added x concentration added
= 5 uL x 500ug/mL = 5uL x 500ug/ 1000uL = 2.5ug
similarly, for the next ones, amount of protein added can be calculated; they will be 5 ug, 10ug, 25ug, 50 ug
b. concentration of standard sample = amount protein added / volume of sample
= 2.5ug / (5uL + 995uL) = 2.5ug/ I000uL = 2.5ug/mL
Similarly, concentrations of the next ones can be calculated; the will be 5ug/mL, 10 ug/mL, 25 ug/mL, 50 ug/mL
c.
Paste this table in excel, select all the cells and then choose scatter (x-y) plot from the plethora of graphs.
You will get the STANDARD CURVE (graph) with absorbance in Y axis and Concentration of proteins in X axis.
d. Equation of the line: Y= 0.0325X
Y- absorbance; X - [Protein]
for unknown protein, the equation becomes: 0.132= 0.0325 [protein]
hence, [protein]= 0.132/0.0325 = 4.06 ug/mL
e. original concentration = [protein] from (d) x 1000/5 (I000uL in dultion / 5uL of stock)
= 4.06 x 1000/5 = 812 ug/mL
Concentration of proteins(ug/ mL) Absorbance 0 0 2.5 0.082 5 0.166 10 0.314 25 0.814 50 1.622Related Questions
Navigate
Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.