Protein Assay

Protein Assay
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Protein Assay
Purpose
The purpose of this lab exercise was to determine the protein concentration of solutions by the Lowry and Bradford Assays.
Materials and Methods/Procedure
Materials
Materials include a spectrophotometer, vortex mixer, cuvettes, micropipettes, marker pen, water baskets, distilled water and micropipette tips. They also include Bovine Serum Albumin (BSA) standard solution, Sodium Phosphate buffer, Lowry reagents A and B, Bio-Rad Dye reagent solution and BSA solution of unknown concentrations.
Methods
Part I. Lowry Assay. A series of 6 Eppendorf tubes containing a wide range of the protein standard solution was prepared to a final volume of 25uL and labeled. A control tube containing 25uL of water was also added. A series of tubes ranging from 0 to 5 to 40ug BSA were made. This was followed by preparation of a series of dilutions with unknown concentration by adding water in different volumes to a final 25uL. A 125uL Reagent A was added to each microfuge tube and mixed thoroughly with a vortex, followed by the addition of 1000uL of Reagent B to each tube and an immediate and thorough vortex. The solution was incubated for 15 minutes and absorbance’s read at 650nm after blanking the spectrophotometer with tube #1.
Part II.A 760uL solution of 0.01mg/L BSA standard was prepared by diluting a 2.0mg/L of the standard solution with 10mM NaH2PO4 AT pH 7.0. This was followed by the preparation of standard serial solutions with a final volume of 800uL.

A blank tube containing 800uL of water was used as control. A set of six tubes with varying dilutions was then prepared while unknown + water was kept at 800uL. A 20uL of Bio-Rad Dye Agent was added to both standards and unknowns followed by an immediate vortex. Tubes were incubated at room temperature for 10 minutes. Thereafter, the absorbance of each sample was measured on the spectrophotometer by setting the wavelength at 595 nm after blanking using tube labeled #7.
Results and Analysis
Part I. Lowry Assay

Table 1.0. Lowry Assay (1-6)
Tube # 1 2 3 4 5 6
Vol. of 2.00 mg/mL BSA(µL) 0 2.5 5 10 25 20
Vol. of H20 (µL) 25.0 22.5 20 15 10 5
Vol.of Reagent A (µL) 125. 125. 125. 125. 125. 125.
Vol.of Reagent B (µL) 1000 1000 1000 1000 1000 1000
Amount of BSA (µg) 0 5.00 10.0 20.0 30.0 40.0
Absorbance of 650 nm, A650 0.000 0.051 0.111 0.196 0.280 0.347
Table 1.1. Lowry Assay (7-12)
Tube # 7 8 9 10 11 12
Vol.of Unknown(µL) 2.50 5.00 10.0 15.0 20.0 25.0
H20(µL) 22.5 20 15 10 5 10
Vol.of Reagent A (µL) 125. 125. 125. 125. 125. 125.
Vol. of Reagent B (µL) 1000 1000 1000 1000 1000 1000
Absorbance at 650 nm, A650 0.008 0.022 0.0477 0.071 0.097 0.106
Amount of BSA (µg) -0.367 1.24 4.11 6.87 9.86 10.89
Concentration of BSA (mg/ml) -0.147 0.248 0.411 0.458 0.493 0.435
The BSA Concentration (mg/ml) or final protein concentration values are obtained using the dilution equation;
M1V1=M2V2Where M1 and M2 are initial and final concentration
V1 and V2 are initial and final concentration; Dilution factor=V2/V1Fig 1. Standard Curve for Lowry Assay

The unknown amounts Amount of BSA (ug) is obtained from the graph sing the linear equation; y=mx+c. The best line of fit is obtained at absorbance 0.12+0.272=0.195 which corresponds to 20 ug/ml of the substance. The gradient (m) of the line becomes;
0.19520=0.098The gradient estimates the absorbance coefficient for BCA reagent. It helps in calculating the concentration of the unknown from the absorbance using the relation;
Concentrationc=(Absorbance(Abs))/Absorbance coefficient(m) For tube 7, the value is 0.0080.098=0.082The subsequent values are obtained as; 0.224, 0.487, 0.724, 0.990, 1.082
The mean value = 3.589/6 = 0.598
Standard deviation is given by the relation; =xi-m2n-1It is obtained as; (1.327/5)= 0.515Therefore the concentration of the unknown according to Lowry assay is given as;
0.598 + or -0.515 ug/ml
Part II. Bradford Assay
The amount(uL) of BSA needed from the 2.0mg/L BSA standard needed to prepare 760 uL of 0.1mg/L is determined using the dilution equation as follows;
M1V1=M2V2M1=760uL*0.1mgL-12.0mgL-1=38uLThe amount of buffer (NaH2PO4) needed to make up the new solution is given as;
716-38=722uLTable 2.0. Bradford Assay (1-6)
Tube# 1 2 3 4 5 6
Vol. of 0.1 mg/mL BSA(µL) 0 10 20 40 60 80
Vol. of H20 (µL) 800 790 780 760 740 720
Vol. of Bio-rad Dye Reagent (µL) 200. 200. 200. 200. 200. 200.
Amount of BSA (µg) 0 1.00 2.00 4.00 6.00 8.00
Absorbance at 596 nm, A595 0.000 0.077 0.114 0.209 0.269 0.327
Table 2.1. Bradford Assay (7-12)
Tube# 7 8 9 10 11 12
Vol of unknown(µL) 10.0 20.0 50.0 75.0 100.0 150.0
Vol. of H20(µL) 790 780 750 725 700 650
Vol. of Bio-Rad Dye Reagent(µL) 200. 200. 200. 200. 200. 200.
Absorbance at 595 nm, A595 0.087 0.154 0.509 0.822 0.972 1.546
Amount of BSA (µg) 1.33 3.06 12.2 20.32 24.2 39.03
Concentration of BSA (mg/ml) 0.133 0.153 0.244 0.271 0.242 0.26
The BSA Concentration (mg/ml) or final protein concentration values are obtained using the dilution equation; M1V1=M2V2Fig2. Standard Curve for Bradford Assay

The best line of fit is obtained at the center of absorbance between 0.21+0.272=0.24 which corresponds to 5.2 ug/ml of the substance. The gradient (m) of the line becomes;
0.245.2=0.046, which is the absorbance coefficient for BCA reagent
Using the relation; C=Abs/m) ,
For tube 7, the value is 0.0870.046=1.891The subsequent values are obtained as; 3.348, 11.065, 17.87, 20.04, 33.61
The mean value = 87.824/6 = 14.64
The standard deviation is given as; (361.426/5)= 8.5Therefore the concentration of the unknown according to Bradford Microassay is given as;
14.64 + or – 8.5 ug/ml.
Questions
1. Goals of the experiment
The main aim of this lab exercise was to determine the unknown protein concentration.
2. Key Theoretical principles and their importance to the experiment
Graphical analysis is used to determine the unknown concentrations by making comparisons using the standard curve. The experiment uses the technique of serial solutions. One dilution is made then part of the first dilution is used to make a more dilute solution (Olson, 3). Blanking the spectrophotometer helps in subtracting all color which is not based on the protein
3. Key Equations and Terms Involved
The main equation used in the experiment is the dilution equation. The equation is given as follows;
M1V1=M2V2 or M1V1=M2V2 or V1/V2=M2/M1Where M1 and M2 are initial and final concentration
V1 and V2 are initial and final concentration
This equation is also used to calculate the dilution factor which is given as;
Dilution factor=final dilute solution volume(V2)/(Volume from stock solutionV1 The linear equation; y=mx+c which is used in graphical analysis when determining unknown concentrations.
The standard deviation equation is used to map the extent in which data deviated from the mean value. The equation is given as;
S=(xi-m2n-1Where xi are values of the data, m is the mean value and n is the number of data points.
4. Key Instrumentation
Spectrophotometer is a fundamental instrument in the experiment where it is used to measure absorbance of the unknown solutions. Mixing the protein with an assay reagent forms a complex which absorbs light under a certain which is measured by the spectrophotometer at a certain wavelength.
Discussion and Conclusion
The data and analysis show that Bradford Assay is the most reliable for the unknown proteins. The obtained concentration is more tangible than the error. Bradford assay also produced higher values for protein. Errors during the experiment could be caused by poor mixing. The exercise has confirmed that the two reliable ways of measuring protein concentration. The standard deviation also indicates that better controls should be incorporated into the experiment.

Works Cited
Olson, Bradley JSC, and John Markwell. “Assays for determination of protein concentration.” Current protocols in protein science (2007): 3-4.