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Lecture

Determinationprotconcen.docx

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Department
Biology
Course
BIOL 2324
Professor
Thomas Manning
Semester
Fall

Description
Determination of Protein Concentration Melissa Johnson Manasa Madasu Mon 2:50 Results Part A: Safronin 0 Solution mL soln. #1 mL Dist. H20 Final Conc. (mg/L) 1 ­ ­ 10 2 30 20 6 3 25 25 5 4 20 30 4 5 10 40 2 6 1 49 0.2 Table 1. Concentrations of dilutions of Safranin 0.   Calculations1 1 V2=2 V Soln 1: (1mg/mL) (1mL) = x (100mL)  ▯0.01mg/mL * 1000 = 10mg/L Soln 2: (10mg/L) (30mL) = x (50mL)  ▯6mg/L Soln 3: (10mg/L) (25mL) = x (50mL)  ▯5mg/L Soln 4: (10mg/L) (20mL) = x (50mL)  ▯4mg/L Soln 5: (10mg/L) (10mL) = x (50mL)  ▯2mg/L Soln 6: (10mg/L) (1mL) = x (50mL)  ▯0.2mg/L nm 400 425 450 475 500 525 550 575 600 625 650 Absorbance(A) 0.027 0.064 0.144 0.328 0.608 0.700 0.297 0.064 0.014 ­0.005 ­0.003 Table 2. Absorption spectrum values of Safranin 0. Figure 1. Graph of Safranin 0 Absorption Spectrum Table 3. Standard curve values for Safranin 0 and unknown dilution absorbances at  525nm. Safranin 0 Unkown Solution 1 2 3 4 5 6 1:10 1:100 1:1000 Conc. (mg/L) 10 6 5 4 2 0.2 X X X Absorbance  1.18 0.70 0.55 0.43 0.18 0.04 >3 2.146 0.265 (A) 5 1 9 8 9 3 Figure 2. Standard curve of Safranin 0 and unkown point plotted at 0.265.   Concentration of unknown at a dilution of 1:1000: y= 0.1159x  ▯y= 0.265  ▯0.265 =  0.1159x  ▯x= 2.2865 Final concentration of stock solution: 2.2865 * 1000 = 2286.5mg/L Table 4. Estimated concentrations of unknown stock solution and dilutions. Unkown Solution Stock 1:10 1:100 1:1000 Estimated Conc.  2286. >12 >12 2.2865 (mg/L) 5 Absorbance (A) N/A >3 2.146 0.265 Part B: Lowry Assay Table 5. Lowry Assay standard (bovine serum albumin) absorbances and unknown  protein. Bovine Serum Albumin (BSA) Unkown Conc. (mg/L) 20 50 100 150 200 500 Stoc 1:10 1:100 k Absorbance  0.08 0.08 0.119 0.24 0.32 0.89 0.48 0.04 0.003 (A) 8 2 8 2 0 5 0 Figure 3. Standard Curve for Lowry Assay standards. Concentration of unknown:  stock: y= 0.0017x  ▯y= 0.485  ▯0.485= 0.0017x  ▯x= 285.29mg/L  1:10: 0.040= 0.0017x  ▯x= 23.53mg/L  1:100: 0.003= 0.0017x  ▯x= 1.77mg/L Table 6. Estimated Concentrations of unknown protein.  Unkown Solution Stock 1:10 1:100 Estimated Conc.  285.29 23.53 1.77 (mg/L) Absorbance (A) 0.485 0.040 0.003 Ave conc. of stock soln. (23.53*(10) + 1.77*(100) +  285.29) /3 = 232.53mg/L Discussion We performed the absorption spectrum to find the wavelength at which the  absorbance of Safranin 0 was the greatest at a given concentration.  We did this to give  more accurate readings for the standard curve of the known and unknown.  The Beer­ Lambert law gives a straight­line relationship between absorbance and concentration, the  slope of which is greater at the highest absorbance of the substance, giving more accurate  final measures.  The standard curves generated from the Beer­Lambert law yield a  straight line relationship of a
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