Practical Pharmaceutical Techniques
Essay by Warda Sahal • March 24, 2018 • Lab Report • 530 Words (3 Pages) • 1,178 Views
1609086
WARDA SAHAL
5PY015
PRACTICAL PHARMACEUTICAL TECHNIQUES
BLOCK 1: PROTEIN ANALYSIS
Experiment 1: Protein assay using the “Bradford” method
Abstract:
Materials and Methods:
Refer to practical schedule.
Results and Calculations:
PART 1:
Volume used (ml) | ||||||||
Tube number | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
Standard protein | 0 | 0.02 (20µl) | 0.04 (40µl) | 0.06 (60µl) | 0.08 (80µ) | 0.12 (120µl) | 0.16 (160µl) | 0.2 (200µl) |
Water | 0.8 (800 µl) | 0.78 (780µl) | 0.76 (760µl) | 0.74 (740µl) | 0.72 (720µl) | 0.68 (680µl) | 0.64 (640µl) | 0.6 (600µl) |
Bradford solution | 0.2 (200µl) | 0.2 (200µl) | 0.2 (200µl) | 0.2 (200µl) | 0.2 (200µl) | 0.2 (200µl) | 0.2 (200µl) | 0.2 (200µl) |
Absorbance at 595nm | 0 | 0.051 | 0.071 | 0.129 | 0.166 | 0.252 | 0.323 | 0.413 |
µ protein/test | 0 | 1 | 2 | 3 | 4 | 6 | 8 | 10 |
Table 1: Calculating the quantity/test (µg/test) using a standard protein solution of known concentration
The following equation was used to calculate the quantity of protein (µg/test):
[pic 1]
For example, the calculations for the quantity/test for tube number 3 are as follows:
Quantity/test = 50 µg/ml x 0.04 ml
Quantity/test = 2 µg/test
The results in table 1 were used to prepare diagram 1, ‘Standard Calibration Curve 1’ below:[pic 2]
The gradient of the line on the graph can be calculated using the following equation:[pic 3]
[pic 4]
m = 0.325 – 0.08 / 8 – 2
m = 0.041 Absorbance at 595nm per µg protein
PART 2:
Tube | 1 | 2 | 3 | 4 | 5 |
Dilution factor | 1:2 | 1:4 | 1:8 | 1:16 | 1:32 |
Unknown protein (µl) | 200 | 200 of tube 1 | 200 of tube 2 | 200 of tube 3 | 200 of tube 4 |
Water (µl) | 200 | 200 | 200 | 200 | 200 |
A595 | *0.150[pic 5] | 0.085 | 0.042 | 0.026 | 0.020 |
Table 2: Absorbance values obtained from the diluted samples of the unknown protein
* This absorbance value is the most suitable as it is ½ way between the minimum and the maximum absorbance values from the ‘Standard Calibration Curve 1’.
PART 3:
Volume used (ml) | ||||||||
Solution used (µl) | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
Unknown protein | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 |
Water | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 |
Bradford solution | 200 | 200 | 200 | 200 | 200 | 200 | 200 | 200 |
Absorbance at 595nm | 0.271 | 0.266 | 0.265 | 0.265 | 0.269 | 0.261 | 0.272 | 0.263 |
*µ protein/test | 6.56 | 6.44 | 6.42 | 6.42 | 6.51 | 6.32 | 6.59 | 6.37 |
Table 3: Calculating the quantity (in µg) of protein in each of cuvettes 1 – 8
* These values were calculated using the y = mx equation where y = A595, m is the gradient which was 0.0413 and x is the unknown. With cuvette 1, the calculations are as follows:
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