Practical Pharmaceutical Techniques
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1609086WARDA SAHAL5PY015PRACTICAL PHARMACEUTICAL TECHNIQUESBLOCK 1: PROTEIN ANALYSISExperiment 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 12345678Standard protein00.02 (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 solution0.2 (200µl)0.2 (200µl)0.2 (200µl)0.2 (200µl)0.2 (200µl)0.2 (200µl)0.2 (200µl)0.2 (200µl)Absorbance at 595nm00.0510.0710.1290.1660.2520.3230.413µ protein/test012346810Table 1: Calculating the quantity/test (µg/test) using a standard protein solution of known concentrationThe 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 mlQuantity/test = 2 µg/testThe 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 – 2m = 0.041 Absorbance at 595nm per µg protein PART 2:Tube12345Dilution factor1:21:41:81:161:32Unknown protein (µl)200200 of tube 1200 of tube 2200 of tube 3200 of tube 4Water (µl)200200200200200A595*0.150[pic 5]0.0850.0420.0260.020Table 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)12345678Unknown protein100100100100100100100100Water 100100100100100100100100Bradford solution200200200200200200200200Absorbance at 595nm0.2710.2660.2650.2650.2690.2610.2720.263*µ protein/test6.566.446.426.426.516.326.596.37Table 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: