Protein Quantification

Topics: Bradford protein assay, Assay, Standard deviation Pages: 5 (1458 words) Published: April 3, 2013
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 their aggregate protein concentration values.

Table 2: Concentration of protein values, mean, standard deviation, and coefficient of variation percentage (respectively shown) for three trials of the BSA Standard samples. Sample| Absorbance ( 595 nm )| Dilution Factor| Protein Concentration ( mg/mL )| Mean (mX)| St Dev(S)| % CV| BSA Std.Rep 1| 0.566| 1| 0.291| 0.33| 0.05| 15.87|

BSA Std. Rep 2| 0.732| 1| 0.393| | | |
BSA Std. Rep 3| 0.608| 1| 0.317| | | |

Table 3: Concentration of protein values, mean, standard deviation, and coefficient of variation percentage (respectively shown) for three trials of the Unknown Pepsin samples. Sample| Absorbance ( 595 nm )| Dilution Factor| Protein Concentration ( mg/mL )| Mean (mX)| St Dev(S)| % CV| Pepsin UnknownRep 1| 0.051| 1| -0.028 *| -0.04 *| 0.02 *| 54.05 *| Pepsin Unknown Rep 2| 0.060| 1| -0.023 *| | | |

Pepsin Unknown Rep 3| 0.000| 1| -0.060 *| | | |
* NOTE: These negative protein concentration values yield experimentally impossible results. As such, one assumes negative concentration values (mg/mL) as zero. The coefficient variation is thus 0%.

Table 4: Concentration of protein values, mean, standard deviation, and coefficient of variation percentage (respectively shown) for three trials of the Unknown Lysozyme samples. Sample| Absorbance ( 595 nm )| Dilution Factor| Protein Concentration ( mg/mL )| Mean (mX)| St Dev(S)| % CV| Lysozyme UnknownRep 1| 0.564| 1| 0.289| 0.29| 0.01| 1.03| Lysozyme Unknown Rep 2| 0.570| 1| 0.293| | | |

Lysozyme Unknown Rep 3| 0.562| 1| 0.288| | | |

Table 5: Concentration of protein values, mean, standard deviation, and coefficient of variation percentage (respectively shown) for three trials of unknown protein samples. Sample| Absorbance ( 595 nm )| Dilution Factor| Protein Concentration ( mg/mL )| Mean (mX)| St Dev(S)| % CV| UnknownRep 1| 0.488| 1| 0.242| 0.24| 0.01| 3.28...

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
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