Bradford Method for Determination of Protein Concentration

Cuntapay, Giormaru, Dela Cruz, Katrina, Dela Cruz, Patrixia Keith, Delloro, Sean Willy, Dimalanta, Ralph Kevin

Abstract

Quantitative determination is essential in many fields of protein study. It depends on the nature of the protein, nature of other components in the protein sample, desired speed, accuracy and sensitivity. One method for determination of protein concentration is Bradford protein assay. It is a spectroscopic analytical procedure used to measure the concentration of protein in a solution. This procedure relies on the binding of the dye Coomassie Brilliant Blue G250 to protein, in which the dye is proportional to the protein concentration. In this experiment, blank and 3 other test tube was assigned with a certain volume of Bovine albumin standard and a volume of distilled water. The fourth test tube will constitute the 1.5 mL of unknown protein. The absorbance was measured using the Ultravioletvisible spectrophotometer and the protein concentration was computed using the dilution equation. The protein concentration of the unknown was determined by using two methods: linear regression method and graphical method

Introduction
A rapid and accurate method for the estimation of protein concentration is essential in many fields of protein study. Quantitative determinations of proteins can be titrimetricelemental, gravimetric or spectroscopic. An assay originally described by Bradford has become the preferred method for quantifying protein in many laboratories. This spectroscopic technique is simpler, faster, and more sensitive than the Lowry method. Moreover, when compared with the Lowry method, it is subject to less interference by common reagents and nonprotein components of biological samples. The Bradford assay relies on the binding of the dye Coomassie Brilliant Blue G250 to protein, in which the dye is proportional to the protein concentration. Without the protein, the solution is red-brown in its acidic solution and when protein binds, the pKa of the dye shifts causing the dye to become blue. Detailed studies indicate that the free dye can exist in four different ionic forms for which the pKa values are 1.15, 1.82, and 12.4. Of the three charged forms of the dye that predominate in the acidic assay reagent solution, the more cationic red and green forms have absorbance maxima at 470 nm and 650 nm, respectively. In contrast, the more anionic blue form of the dye, which binds to protein, has an absorbance maximum at 590 nm. The assay is sensitive to about 20 to 200 g protein. Thus, the quantity of protein can be estimated by determining the amount of dye in the blue ionic form. This is usually achieved by measuring the absorbance of the solution at 595 nm. However, there are also disadvantages when using this method. The Bradford assay is inhibited by the presence of detergents. For example, Sodium dodecyl sulfate (SDS), a common detergent, may be found in protein extracts because it is used to lyse cells by disrupting the membrane lipid bilayer. While other detergents interfere with the assay at high

concentration, the interference caused by SDS is of two different modes, and each occurs at a different concentration. When SDS concentrations are below critical micelle concentration (known as CMC, 0.00333%W/V to 0.0667%) in a Coomassie dye solution, the detergent tends to bind strongly with the protein, inhibiting the protein binding sites for the dye reagent. This can cause underestimations of protein concentration in solution. When SDS concentrations are above CMC, the detergent associates strongly with the green form of the Coomassie dye, causing the equilibrium to shift, thereby producing more of the blue form. This causes an increase in the absorbance at 595 nm independent of protein presence. Lastly, it is also linear over a short range, typically from 0 g/ml to 2000 g/ml, often making dilutions of a sample necessary before analysis.

Experimental Procedure
In this experiment, Bovine serum albumin (BSA) of 100 g/ml concentration was used as the standard protein. To determine the protein concentration of an unknown sample, 5 fold dilutions was used to guarantee that the protein concentration was within the range of the assay. Also, a blank or the test tube without the standard protein and an unknown protein was used. The series of test tubes were prepared as follows: Table 1. Test Tube Preparations Volume Standard (mL) 0 0.1 0.5 1 1.5 Volume Water (mL) 1.5 1.4 1 0.5 0

Tube No. Blank 1 2 3 Unknown

In each test tube, 1.5 mL of Bradford reagent which composed of Coomasie Brilliant Blue G-250 dye, 95% ethanol and 85% phosphoric acid, was added. The solution was mixed well by inversion or gentle vortex-mixing. To measure the absorbance, UV-VIS Spectrometer was used. First, each solution was placed in separate cuvettes. The blank or the tube containing 100 g standard should give an A595 value of about 0.4.The absorbance of the samples and standards against the reagent blank was measured between 2 minutes and 1 hour after mixing. The albumin standard curve was constructed by plotting A595 against protein concentration (g/mL) and the concentration of the unknown was determined graphical method or by linear regression analysis.

native state, consequently exposing its hydrophobic pockets. These pockets on the protein's tertiary structure bind non-covalently to the non-polar region of the dye via van der Waals forces, positioning the positive amine groups in proximity with the negative charge of the dye. The bond is further strengthened by the ionic interaction between the two. The binding of the protein stabilizes the blue form of the Coomassie dye; thus the amount of the complex present in solution is a measure for the protein concentration, and can be estimated by use of an absorbance reading.
PROTEIN Basic and aromatic side chains + =

BLUE

Results and discussions

The Bradford protein assay is a spectroscopic analytical procedure used to measure the concentration of protein in a solution. Particularly, Ultraviolet-Visible spectrophotometer was used to measure the absorbance of the standard and an unknown protein. Primarily, it uses light in the visible and adjacent (near-UV and near-infrared (NIR)) ranges as shown in Figure 1. The absorption in the visible range directly affects the perceived color of the chemicals involved. In this region of the electromagnetic spectrum, molecules undergo electronic transitions.

Figure 2. Chemical Reaction of Coomasie dye with Protein Spectrophotometry was based on BeerLamberts Law which relate the absorbance of a solution to the concentration of a particular solute to in that solution, as shown in the formula in Figure 3.

[ ]
Figure 3. Beer-Lambert Law Where A is the measured absorbance, I0 is the intensity of the incident light at a given wavelength, I is the transmitted intensity, L the pathlength through the sample, and c the concentration of the absorbing species. For each species and wavelength, is a constant known as the molar absorptivity or extinction coefficient. This constant is a fundamental molecular property in a given solvent, at a particular temperature and pressure, and has units of 1 / M * cm or often AU / M * cm. The absorbance and extinction are sometimes defined in terms of the natural logarithm instead of the base-10 logarithm. The Beer-Lambert Law is useful for characterizing many compounds but does not hold as a universal relationship for the concentration and absorption of all substances. lllThus, for a fixed path length, UV-VIS spectroscopy can be used to determine the

Figure 1. Schematic of a wavelength-selectable, single-beam UV-Vis spectrophotometer It is subjective, for example, dependence on the amino acid composition of the measured protein. It is based on an absorbance shift of the dye Coomassie Brilliant Blue G-250 under acid conditions when a redder form of the dye is converted into a bluer form on binding to protein. During the formation of this complex, two types of bond interaction take place, as shown in Figure 2: the red form of Coomassie dye first donates its free electron to the ionizable groups on the protein, which causes a disruption of the protein's

concentration of the absorber in a solution. It is necessary to know how quickly the absorbance changes with concentration. This can be taken from references (tables of molar extinction coefficients), or more accurately, determined from a calibration curve. lllOrganic compounds, especially those with a high degree of conjugation, also absorb light in the UV or visible regions of the electromagnetic spectrum. The solvents for these determinations are often water for water soluble compounds, or ethanol for organic-soluble compounds. (Organic solvents may have significant UV absorption; not all solvents are suitable for use in UV spectroscopy. Ethanol absorbs very weakly at most wavelengths.) Solvent polarity and pH can affect the absorption spectrum of an organic compound. Tyrosine, for example, increases in absorption maxima and molar extinction coefficient when pH increases from 6 to 13 or when solvent polarity decreases. lllThe basic parts of a spectrophotometer are light source, a holder for the sample, a diffraction grating or monochromator to separate the different wavelengths of light, and a detector. The radiation source is often a Tungsten filament (300-2500 nm), a deuterium arc lamp, which is continuous over the ultraviolet region (190-400 nm) or more recently, light emitting diodes (LED) and Xenon arc lamps (Continuous Spectra from 160-2,000nm) for the visible wavelengths. The detector is typically a photodiode or a CCD. Photodiodes are used with monochromators, which filter the light so that only light of a single wavelength reaches the detector. Diffraction gratings are used with CCDs, which collects light of different wavelengths on different pixels. lllThe spectrophotometer was used to determine the absorbance of each solution, shown in Table 2.This instrument measures the intensity of light passing through a sample (I), and compares it to the intensity of light before it passes through the sample (Io). The ratio I / Io is called the transmittance, and is usually expressed as a percentage (%T). The absorbance, A, is based on the transmittance: A = log(%T / 100%). Table 2. Absorbance (A) measured using UV-Vis Spectrophotometer Tube No. Blank 1 2 3 Unknown

In this experiment, the protein concentration of each dilution standard was determined by using the concentration formula as shown in Figure 4,where C1 is the concentration of Bovine serum albumin (BSA) standard (100 g/mL), V1 is the volume of standard protein, C2 is the protein concentration determined in this equation and V2 is the total volume of the solution (1.5 mL).

Figure 4. Dilution Equation The computed protein concentration, C 2, was shown in table 3 below. For the unknown protein, the value of C2 was X. Table 3. Computed Protein Concentrations, C2 Tube No. Blank 1 2 3 Unknown C2(g/mL) 0 6.67 33.33 66.67 X

The concentration of the unknown protein was determined by two ways: first, by graphing the computed amount of protein concentration against absorbance and by using linear regression method. Through graphical method the concentration of the unknown protein was determined, as shown in Figure 5, the protein concentration on the x-axis while absorbance was in the y-axis.

Unknown

3 2

A 0 0.7 0.949 1.037 1.28

blank

Figure 5. Protein Standard Curve The best line through the points was drawn because the protein standard curve was not

linear due to errors in experiments. The line was not necessarily the best line through the origin and the other points. For the unknown protein, the concentration was determined by tracing the point in which the absorbance, 1.280, met the best-fit line of the graph. The concentration was approximately measured to be 77 g/mL. lllOn the other hand, for a more accurate result the linear regression method was used to determine the protein concentration. The equation used was shown in Figure 6, where y is the value of the absorbance, m is the slope, and b is the y-intercept.

Noble, J.E.; Bailey, M.J.A. (2009). Methods Enzymol. Quantitation of Protein. Retrieved fromohttp://en.wikipedia.org/wiki/Bradford _protein_assay Zuo, S.-S. and Lundahl, P. (2000). Analyt. Biochem. A micro-Bradford membrane protein assay. 284, 162164.

Figure 6. Linear Equation Using a calculator, the values of slope and yintercept was determined with the use of the known x, protein concentration of the standard, and y, absorbance. The value for m is 0.0122, b is 0.3443 and y is 1.280. The computation as shown in Figure 6 was used to determine protein concentration of the unknown, 76.69 g/mL. The protein concentration using the graphical and linear regression method was almost the same.

Figure 7. Computation for Protein Concentration using Linear Equation The protein concentration using the graphical and linear regression method was almost the same.

References
Bradford, M. M. (2000). Analyt. Biochem. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. 72,248254. Bradford Method: Colorimetric Protein Assay Retrievedofromphttp://bioteachnology.com/ protein/bradford-method-colorimetricprotein-assay Crisostomo, A. et. al(2010). Laboratory Manual in General Biochemistry. Isolation and Characterization of Proteins. South Triangle, Quezon City: C&E Publishing, Inc. Ultraviolet-visible spectrophotometry. Retrieved fromohttp://en.wikipedia.org/wiki/Ultraviole t%E2%80%93visible_spectroscopy