Lab 1: Determination of Protein Concentration

The purpose of this investigation is to learn to use spectrophotometer as means of

measuring the protein and dye concentrations in various samples at different dilutions.

Results
Part A. Safranin O

Table1. Concentrations of Dilutions of Safranin O

Solution mL Solution #1 mL D.H20 FinalConc.(mg/ml)

1 1 ml 99 ml 10 mg/ml
2 30 ml 20 ml 6 mg/ml
3 25 ml 25 ml 5 mg/ml
4 20 ml 30 ml 4 mg/ml
5 10 ml 40 ml 2 mg/ml
6 1 ml 49 ml 0.2 mg/ml

1) Calculations: C1V1 = C2V2

Solution 1: 1000 mg * 1 ml = 100 ml * X mg = 1000 mg / 100 mg = 10 mg/ml
Solution 2: 10 mg/ml * 30 ml = 50 ml * X mg = 300 mg / 50 ml = 6 mg/ml
Solution 3: 10 mg/ml * 25 ml = 50 ml * X mg = 250 mg / 50 ml = 5 mg/ml
Solution 4: 10 mg/ml * 20 ml = 50 ml * X mg = 200 mg / 50 ml = 4 mg/ml
Solution 5: 10 mg/ml * 10 ml = 50 ml * X mg = 100 mg / 50 ml = 2 mg/ml
Solution 6: 10 mg/ml * 1 ml = 50 ml * X mg = 10 mg / 50 ml = 0.2 mg/ml

Table 2. Absorption Spectrum of Safranin O

Nm 400 425 450 475 500 525 550 575 600 625 650
Absorbance 0.026 0.071 0.166 0.358 0.612 0.676 0.256 0.043 0.008 0.002 0.001

The absorbance at which the standards and the unknowns will be run is 525 nanometers
because at this wavelength the absorbance was the highest.
Table 3. Standard Curve of Safranin O
UNKNOWNS
Solution 1 2 3 4 5 6 1/10 1/100 1/1000
Absorbance(A) 1.064 0.668 0.533 0.421 0.219 0.028 3 1.335 0.169
Conc.(mg/ml) 10 6 5 4 2 0.2 280.3 1244.9 1542.1
Calculations:
(3 – 0.004) / 0.107 = 28 * 10 = 280.3 mg/ml
(1.335 – 0.004) / 0.107 = 12.449 * 100 = 1244.9 mg/ml
(0.169 – 0.004) / 0.107 = 1.5421 * 1000 = 1542.1 mg/ml
The unknown concentration is 1542.1 mg/ml. The 1:1000 dilution was used because the
absorbance acquired was within the range of the standard curve and the other two
dilutions were way above the maximum value of the standard curve.
Part B. Lowry Assay

Table 1. Absorbance of Lowry Standards

Standard 1(Blank1:5) 2 3 4 5 6 7
Concentration 91.67 20 50 100 150 200 500
Absorbance 0.057 0.137 0.196 0.347 0.461 0.552 0.885

The samples were read at 660 nm.

Table 2. Unknown Concentrations

Concentration 1 1 / 10 1 / 100
Absorbance 0.807 0.089 0.011
The absorbance value at the concentration 1 is used because it is high enough for good
detection and fits within the standard curve range. The other two are shown, but were not
used because the absorbance was too low to be valid.
Calculations: (0.807A – 0.1383A) / 0.0016 = 417.94 mg/ml
((0.089A*10) – 0.1383A) / 0.0016 = 469.81 mg/ml
((0.011A*100) – 0.1383A)/0.0016 = 601.12 mg/ml
(0.057 A * 5) – 0.1383A)/0.0016 = 91.67 mg/ml (The Sample Blank value)
The unknown concentration is 471.94 mg/ml
Discussion Questions
The reasoning behind qunatitating the absorption spectrum of Safranin O was to find the
wavelength, which would produce precise measurement of absorbance. The reason why
we chose the maximum wavelength to read the standards and the unknowns is because at
this maximum wavelength was the one at which most absorbance occurred, thus giving
the highest possible result. The standard curve is the relationship between the known
concentrations against which one can test the unknown sample. Knowing the standard
curve values one can acquire the value for the unknown sample. We calculated the
unknown concentrations through the linear relationship formula acquired from the
standard curve. The sample blank was also tested to show the possible amount of error to
be subtracted from the sample read outs. The y value is the absorbance and we were
looking for x, which is the concentration. From the absorbance we subtract the value
where the y value hits the curve and then divide by the slope, thus extrapolating the
values. That would give us the unknown concentration estimate.
The Lowry assay works is by using the Spectrophotometer to quantify the absorbance at
660 nm of the blue solution. The formation of the blue color results from reduction of the
Folin phenol by amino acid tyrosine in the presence of copper. The major difference
between this method and the Biuret and Bradford assays is the fact that Lowry is not
equally sensitive to all proteins. During this experiment all reagents must be kept constant
because the reactions occurring during the experiment would produce the best possible
outcome for concentration with the least amount of error and variable conditions.

Questions

1) Since the solution turns blue in color, it means that it is the color, which is being
refracted, thus suggesting that all other visible wavelengths would be absorbed.

2) If the blue dye had been added to the Safranin O, the readings would be either
same or very close and the relationship would still be linear. Since 2 colors are
absorbed at different wavelengths, even if they are in the same solution, 2
different wavelengths, except for red and blue wavelengths, could be used to
measure their concentrations.

3) 5 g / 1L = 3 g / X L
X = 3/5 L = 0.6 L or 600 mL is needed to make the appropriate concentration

4) Beer – Lambert’s Law is used Absorbance = elc

In this case the e and c are constant, so they can be dropped. We are left with
Absorbance = l (length of a cuvette)
0.05(Cuvette A) = 1 cm
X(Cuvette B) = 1.1 cm
X Cuvette B = 0.05 * 1.1 cm =0.055 absorbance in a cuvette B.