Protein Quantification - BCA Assay Method - Lab Report Example

PROTEIN QUANTIFICATION By: Presented BCA assay method: Reagents: BCA reagent Solutions: Solution A g sodium bicinchoninate (BCA) 2 g sodium carbonate 0.16 g sodium tartrate 0.4 g NaOH 0.95 g sodium bicarbonate up to 100 ml with purified water pH adjusted to11.25 with 10 M NaOH Solution B: 0.4 g cupric sulphate pentahydrate in 10 ml purified water. BCA reagent: 100 volumes of solution A to 2 volumes Procedure: Required: The Protein standard solutions D,E,F,G and H, 18 microcentrifuge tubes, blank(water), three unknown protein concentrations X, Y and Z, floating rack. The assays are carried out in test tubes in duplicates and duplicate reagent blanks containing water instead of protein is ran. The microcentrifuge tubes are labeled appropriately and placed on the floating rack. To each labeled microcentrifuge tube 50 μl of each different protein standard (D,E,F,G and H) or water (for reagent blanks) or unknown samples X, Y and Z are added. 1 ml of the BCA reagent is added,and mixed well by inverting the capped tubes.the rack is transferred to a 60oc water bath and Incubated for 30 minutes. After the 30 minutes of incubation the rank is removed from the water bath and the samples allowed to cool.1 ml of each reaction is transfered to a 1 ml cuvette and A562 in a spectrophotometer read. The spectrophotometer zero is set to using one of the reagent blanks (reaction set up with water). RESULTS: Table 1: Calibration curve of protein standard solutions in BCA assay method γ- globulin concentration (mg/ml) A562 First set A562 Second set WATER 0 0 0 D 1 1.3 1.4 E 0.5 0.902 1.027 F 0.250 0.376 0.426 G 0.125 0.225 0.384 H 0.065 0.098 0.083 Calibration curve for BSA for other group BSA concentration (mg/ml) A562 First set A562 Second set WATER 0 0 0 D 1 1.643 1.669 E 0.5 1.165 1.190 F 0.250 0.640 0.672 G 0.125 0.317 0.344 H 0.065 0.148 0.164 Table 1.1: Calibration curve of unknown protein samples in γ-globulin assay method Sample A562 First set A562 Second set Average Concentration (mg/ml) X 1.408 1.408 1.408 0.949 Y 1.012 1.225 1.118 0.7537 Z 0.090 0.099 0.0945 0.0637 Calibration curve of unknown protein samples in BSA assay method Sample A562 First set A562 Second set Average Concentration (mg/ml) X 1.702 1.716 1.709 0.9255 Y 1.349 1.340 1.345 0.7284 Z 0.053 0.039 0.046 0.0249 Standard Curve using BCA Assay with BSA standard curve and y-globulin curve γ- globulin test BSA test Calculations: Coordinates: when x=0,y=0 and when x=1, y=1.4834. M1 The equation regression line(y-globulin standard curve): Coordinates: (0,0), (1,1.4834) M1= = when x=0,y=0 and when x=1, y=1.8465. M2= The equation regression line(BSA standard curve): Coordinates: (0,0), (1,1.8465) M1= = Calculate the Concentration of unknown protein samples in BSA standard:  mg/ml mg/ml Calculate the Concentration of unknown protein samples in -Globulin standard. 2.Bradford Protein Determination Method: Reagent: Bradford reagent Solutions: Bradford reagent: 100 mg Brilliant Blue G-250 50 ml 95% Ethanol 100 ml 85% orthophosphoric acid up to 1L with purified water Procedure: For the Bradford Assay the materials needed are only the protein standard C, D, E, F, and G, 18 microcentrifuge tubes, rack, unknown samples X,Y and Z. The assays are carried out in duplicate in microcentrifuge tubes. Duplicate reagent blanks containing water instead of protein are ran. The microcentrifuge tubes are labled appropriately and placed in the provided rack To each labelled test tube 20 μl of each different protein standard (C, D, E, F and G) or water (for reagent blanks) or unknown samples X, Y and Z are added. 1 ml of the Bradford reagent is added and mix well by inverting the tube and Incubated at room temperature for 5 minutes. After the 5 minutes of incubation 1 ml of each reaction is transfered to a 1 ml cuvette and A595 in a spectrophotometer is read. The spectrophotometer is set to zero using one of the reagent blanks (reaction set up with water). RESULTS: Table 2: Calibration curve of protein standard solutions in Bradford assay method γ- globulin concentration (mg/ml) A595 First set A595 Second set WATER 0 0 0 C 2 0.774 0.973 D 1 0.451 0.521 E 0.5 0.208 0.366 F 0.250 0.091 0.086 G 0.125 0.052 0.044 BSA concentration (mg/ml) A595 First set A595 Second set WATER 0 0 0 C 2 1.204 1.167 D 1 0.851 0.851 E 0.5 0.545 0.515 F 0.250 0.312 0.276 G 0.125 0.166 0.233 Table 2.1: Calibration curve of unknown protein samples in γ-globulin assay method Sample A595 First set A595 Second set Average Concentration (mg/ml) X 1.112 1.099 1.105 2.4485 Y 0.536 0.483 0.509 1.1279 Z 0.061 0.051 0.056 0.1241 Calibration curve of unknown protein samples in BSA assay method Sample A595 First set A595 Second set Average Concentration (mg/ml) X 1.458 1.412 1.435 2.1326 Y 0.609 0.554 0.5815 0.8642 Z 0.025 0.037 0.031 0.0461 Standard Curve using Bradford Assay with BSA and Y-globulin standard curve γ- globulin test BSA test Calculations: Coordinates: when x=0,y=0 and when x=2, y=0.9026. M1 The equation regression line(y-globulin standard curve): Coordinates: (0,0), (2,0.9026) M1= = when x=0,y=0 and when x=1, y=1.3458. M2= The equation regression line(BSA standard curve): Coordinates: (0,0), (2,1.3458) M1= = Calculate the Concentration of unknown protein samples in BSA standard:  mg/ml mg/ml Calculate the Concentration of unknown protein samples in -Globulin standard. 3. Protein quantification using the Lowry method: Reagents: Lowry reagent 1: 1 ml solution A 50 ml solution B Lowry reagent 2: 10 ml Folin-Ciocalteu phenol reagent 10 ml water Solutions: Protein solution of unknown concentration (X, Y, Z) Solution A: 0.5 g copper sulphate 1 g sodium citrate up to 100 ml with purified water Solution B: 20 g sodium carbonate 4 g sodium hydroxide up to 1 L purified water Procedure: Required: The Protein standard solutions F,G, H, I and J, 18 glass tubes, blank(water), three unknown protein concentrations X, Y and Z, rack. The assays are carried out in test tubes in duplicates.and duplicate reagent blanks containing water instead of protein is ran. The test tubes are labeled appropriately and placed on the rack. To each labeled test tube 500 μl of each different protein standard (F, G, H, I and J) or water (for reagent blanks) or unknown samples X, Y and Z are added. 2.5 ml of the Lowry reagent 1 is added and mixed well using the vortex mixer and Incubated at room temperature for 10 minutes. After the 10 minutes of incubation 250 μl Lowry reagent 2 are added and mixed well using the vortex mixer and Incubated for 30 minutes at room temperature. After the 30 minutes of incubation 1 ml of each reaction is transfered to a 1 ml cuvette and A750 in a spectrophotometer read.The spectrophotometer zero is set to using one of the reagent blanks (reaction set up with water). RESULTS: Table 3: Calibration curve of protein standard solutions in Lowry assay method γ – globulin concentration (mg/ml) A750 First set A750 Second set WATER 0 0 0 F 250 0.564 0.594 G 125 0.311 0.390 H 65.5 0.122 0.129 I 31.25 0.051 0.055 J 15.7 0.024 0.078 Standard Curve using Lowry Reagent with BSA standard curve and Y-globulin standard curve BSA concentration (mg/ml) A750 First set A750 Second set WATER 0 0 0 F 250 0.642 0.661 G 125 0.350 0.331 H 65.5 0.173 0.170 I 31.25 0.071 0.053 J 15.7 0.030 0.029 Table 3.1: Calibration curve of unknown protein samples in γ-globulin assay method Sample A595 First set A595 Second set Average Concentration (mg/ml) X 1.73 0.087 0.91 379.1667 Y 1.057 1.141 1.1 458.3333 Z 0.095 0.080 0.09 37.5 Calibration curve of unknown protein samples in BSA assay method Sample A595 First set A595 Second set Average Concentration (mg/ml) X 2.133 2.096 2.115 813.4615 Y 1.227 1.245 1.236 475.3846 Z 0.086 0.087 0.0865 33.2692 γ- globulin test BSA test Calculations: Coordinates: when x=0,y=0 and when x=250, y=0.6. M1 The equation regression line(y-globulin standard curve): Coordinates: (0,0), (250,0.6) M1= = when x=0,y=0 and when x=250, y=0.65. M2= The equation regression line(BSA standard curve): Coordinates: (0,0), (250,0.65) M1= = Calculate the Concentration of unknown protein samples in BSA standard: mg/ml mg/ml Calculate the Concentration of unknown protein samples in -Globulin standard. 4. Protein quantification using the Biuret method: Reagents: Biuret reagent: Solutions: Protein solution of unknown concentration (X, Y, Z) Biuret reagent: 9 g sodium potassium tartrate 3 g copper sulphate 5 g potassium iodide up to 400 ml with 0.2 M sodium hydroxide Procedure: Required: The Protein standard solutions A, B,C,D and E, 18 microcentrifuge tubes, blank(water), three unknown protein concentrations X, Y and Z, rack. The assays are carried out in tubes in duplicates.and duplicate reagent blanks containing water instead of protein is ran. The microcentrifuge tubes are labeled appropriately and placed on the rack. To each labeled test tube 100 μl of each different protein standard (A,B,C,D and E) or water (for reagent blanks) or unknown samples X, Y and Z are added. 1 ml of the Biuret reagent is added and mixed well by inverting the tubes and Incubated at room temperature for 20 minutes. After the 30 minutes of incubation 1 ml of each reaction is transfered to a 1 ml cuvette and A550 in a spectrophotometer read. The spectrophotometer zero is set to using one of the reagent blanks (reaction set up with water). Table 4: Calibration curve of protein standard solutions in Biuret assay method γ- globulin concentration (mg/ml) A550 First set A550 Second set WATER 0 0 0 A 8 0.286 0.243 B 4 0.081 0.156 C 2 0.041 0.078 D 1 0.020 0.036 E 0.5 0.017 0.020 BSA concentration (mg/ml) A550 First set A550 Second set WATER 0 0 0 A 8 0.156 0.164 B 4 0.076 0.076 C 2 0.039 0.037 D 1 0.019 0.016 E 0.5 0.009 0.007 Table 3.1: Calibration curve of unknown protein samples in γ-globulin assay method Sample A595 First set A595 Second set Average Concentration (mg/ml) X 0.308 0.295 0.302 9.3789 Y 0.011 0.014 0.013 0.4037 Z 0.003 0.003 0.003 0.0932 Calibration curve of unknown protein samples in BSA assay method Sample A595 First set A595 Second set Average Concentration (mg/ml) X 0.289 0.286 0.288 14.6193 Y 0.010 0.011 0.011 0.5584 Z 0.002 0.002 0.002 0.1015 Standard curve using Biuret Assay with BSA and y-globulin standard curves γ- globulin test BSA test Calculations: Coordinates: when x=0,y=0 and when x=8, y=0.1576. M1 The equation regression line(BSA standard curve): Coordinates: (0,0), (8,0.1576) M1= = when x=0,y=0 and when x=8, y=0.2576. M2= The equation regression line(y-globulin standard curve): Coordinates: (0,0), (8,0.2576) M1= = Calculate the Concentration of unknown protein samples in BSA standard: mg/ml mg/ml Calculate the Concentration of unknown protein samples in -Globulin standard. Sample Real concentration BCA Bradford Lowry Biuret BSA -GLO BSA -GLO BSA -GLO BSA -GLO X 12 0.926 0.949 2.133 2.449 813.46 379.17 14.619 9.379 Y 0.6 0.728 0.7537 0.864 1.128 475.38 458.33 0.558 0.404 Z 0.04 0.0249 0.06 0.046 0.124 33.27 37.5 0.1015 0.0932 Read More