CHAPTER 4

PRESENTATION, ANALYSIS AND INTERPRETATION OF THE STUDY

The results and findings are presented in tabular format to visualize a clearer pattern of the
phenomenon behavior of the data and have better interpretations and analyses based on the
objectives stated in Chapter I.

TOTAL PROTEIN CONCENTRATION and ABSORPTION

The total protein concentration of Cryopreserved and Lyophilized Amniotic Membrane in

full term fetus was determined by the Biuret Method and Bradford method (Plate 14), which are
based on UV-visible spectroscopy. These methods used the natural ability of proteins to absorb
(or scatter) light in the UV-visible region of the electromagnetic spectrum, or they chemically or
physically modify proteins to make them absorb (or scatter) light.
Data
Mass of sample: 9.5 mg
Total Volume of solution: 5mL
Aliquot: 1mL diluted to 5mL

Table 4. Analysis of Protein Concentration and its Absorbency in two-methods.

Percent
Sample mcg Dilute Total Total Mass (%)
Trial Concentration
Absorbance protein factor (mcg) (mg) (mg) of
Protein
I 1.13 56.13 280.63 5 1403.13 1.40 9.5 14.77
1.11 54.93 274.69 5 1373.44 1.37 9.5 14.46
1.112 55.0 275.0 5 1375.0 1.38 9.5 14.47
II 1.11 54.88 274.38 5 1371.88 1.37 9.5 14.44
1.111 54.94 274.69 5 1373.44 1.37 9.5 14.46
1.111 54.94 274.69 5 1373.44 1.37 9.5 14.46
III 1.07 52.38 261.88 5 1309.38 1.31 9.5 13.78
1.069 52.31 261.56 5 1307.81 1.31 9.5 13.76
1.071 52.44 262.19 5 1310.94 1.31 9.5 13.80
Legend: Trials I and II are Bradford method, Trial III is Biuret method.

The presence of protein was seen consistently both in Bradford (Trial I and II) and Biuret
method (Trial III) methods as shown in Table 4. The percentages of protein presence in 9.5 mg
(Mass total sample) of Amniotic Membrane ranges between 14.77 – 14.46 % in Bradford
method, while slightly lower to 13.78 – 13.80 % concentration in Biuret method, respectively.
Bradford method evidently showed higher sensitivity for proteins than Biuret test. However, the
two methods were consistent enough in providing an enough evidence of the qualitative and
quantitative analysis of protein in Amniotic Membrane.

Generation of a standard curve is necessary for the purpose of photometric analysis of

Bradford dye procedure. The table 5 shows the measured standard in the Spectrometer for the
purpose of generating a standard curve. To this end, below are seven (7) standards with defined
concentrations both in table 5 and linear graph forms as seen in table 5 and figure 2 and 3
resepectively.
Table 5. Concentration of Amniotic Membrane protein and its absorbance at 584nm

Standard Absorbance
Concentration
20 0.547
40 0.895
60 1.229
80 1.525
120 1.689
160 1.708
200 1.628

Absorbance vs Concentration
2
Absorbance at 584nm

1.5
1
0.5 Absorance vs
Concentration
0
0 50 100 150 200 250
Concentration of Protein, mcg/mL

Figure 2. Concentration versus Absorbance

Note: The figure above showed level of absorption at a certain wavelength to concentration of protein.

Figure 2 shows a generation of standard curve in which the absorbance of protein

increases when the concentration does. The highest absorption is 1.708 at a standard
concentration of 160 mcg/mL. This implies that the data given in a mass sample of 9.5 mg, the
absorption level is high enough to generate information of its effectivity.
 Calibration Curve
2

Absorbance at 584nm 1.5

y = 0.016x + 0.232

1
Calibration Curve
0.5

0
0 20 40 60 80 100
Concentration of protein, mcg/mL

Figure 3. Calibration Curve

The absorbance at 584nm is plotted against protein concentrations. Figure 3 showed a trend
line presentation based on the collected data given at y=0.016x + 0.232 with a .998 correlation.
Using the linear approximation found in experimental data, the concentration of the Amniotic
Membrane was found.
1.708 = 0.016x + 0.998 in which x = 44.375 ug/mL.

One important factor determining the accuracy of the regression curve is the coefficient
of determination R2 (as shown in figure 4 R2 is 0.998). The closer the R2 value is to 1, the closer
the regression curve overlaps with the actual values measured. The correlation factor for
Bradford method was above .99 which indicates precise reading.