Protein Quantification
BIOL 1F90
Expt. #1: Protein Quantification
Student Name: Carlos Osorio
Student ID: 5220710
Lab Section: 34
Date Experiment Performed: Sept. 26th, 2012
Lab Partners: K. Cloutier J. Yang
ABSTRACT Protein concentration analysis is primarily done through an accepted form commonly referred to as the Bradford Protein Assay. The main purpose of this experiment was to observe and record the various protein samples’ absorbency values through the calibrated readings of a spectrophotometer (595 nm calibration). Using the standard curve equation (y = 1.6147x + 0.0968) derived from determining the regression equation of different protein concentrations of BSA, an accurate form of protein concentrations were obtained for three different given samples, including an unknown. A notable empirical observation found in this experiment is the gradual increase in dark color change (i.e., blue) as the concentration of a protein increased with the inverse proportion of water per reagent added.
MATERIALS AND METHODS Procedure and materials for Experiment #1: Protein Quantification were taken from the BIOL 1F90 Laboratory Manual #1, pages 1-9 (Martin, 2012). A change was made to the protocol for procedure 5 in Part B: Assaying Standards and Samples (page 4). In proceeding with the addition of 3 mL of Bradford Reagent to all six cuvettes, indicator was added to nine cuvettes instead.
RESULTS
Table 1: Recorded absorbency values of five different test tubes with altering protein concentrations containing different volumes of BSA stock for two trials. Sample | Blank | 1 | 2 | 3 | 4 | 5 | Protein( mg/mL ) | 0.0 | 0.2 | 0.4 | 0.6 | 0.8 | 1.0 | Absorbance( 595 nm )Run #1 | 0.000 | 0.570 | 0.796 | 1.140 | 1.580 | 1.760 | Absorbance( 595 nm )Run #2 | 0.000 | 0.488 | 0.710 | 0.985 | 1.240 | 1.580 | Average Absorbance | 0.000 | 0.529 | 0.753 | 1.063 | 1.410 | 1.670 |
Figure 1: Trend showing gradual increase of average absorbencies between two trials in
Expt. #1: Protein Quantification
Student Name: Carlos Osorio
Student ID: 5220710
Lab Section: 34
Date Experiment Performed: Sept. 26th, 2012
Lab Partners: K. Cloutier J. Yang
ABSTRACT Protein concentration analysis is primarily done through an accepted form commonly referred to as the Bradford Protein Assay. The main purpose of this experiment was to observe and record the various protein samples’ absorbency values through the calibrated readings of a spectrophotometer (595 nm calibration). Using the standard curve equation (y = 1.6147x + 0.0968) derived from determining the regression equation of different protein concentrations of BSA, an accurate form of protein concentrations were obtained for three different given samples, including an unknown. A notable empirical observation found in this experiment is the gradual increase in dark color change (i.e., blue) as the concentration of a protein increased with the inverse proportion of water per reagent added.
MATERIALS AND METHODS Procedure and materials for Experiment #1: Protein Quantification were taken from the BIOL 1F90 Laboratory Manual #1, pages 1-9 (Martin, 2012). A change was made to the protocol for procedure 5 in Part B: Assaying Standards and Samples (page 4). In proceeding with the addition of 3 mL of Bradford Reagent to all six cuvettes, indicator was added to nine cuvettes instead.
RESULTS
Table 1: Recorded absorbency values of five different test tubes with altering protein concentrations containing different volumes of BSA stock for two trials. Sample | Blank | 1 | 2 | 3 | 4 | 5 | Protein( mg/mL ) | 0.0 | 0.2 | 0.4 | 0.6 | 0.8 | 1.0 | Absorbance( 595 nm )Run #1 | 0.000 | 0.570 | 0.796 | 1.140 | 1.580 | 1.760 | Absorbance( 595 nm )Run #2 | 0.000 | 0.488 | 0.710 | 0.985 | 1.240 | 1.580 | Average Absorbance | 0.000 | 0.529 | 0.753 | 1.063 | 1.410 | 1.670 |
Figure 1: Trend showing gradual increase of average absorbencies between two trials in
References: Martin, S. (2012) BIOL 1F90 Lab Manual 2012 – 13 Ed. Brock University, St. Catharines, Ontario, Canada. Zor, T.; Selinger, Z. (1996) Linearization of Bradford Protein Assay Increases It’s Sensitivity: Theoretical and Experimental Studies. Analytical Biochemistry, 236: 302-308.v