Protein Assay by the Bradford Method - Lab Report Example

Protein Assay by the Bradford Method Quantitative determination of proteins is essential in many fields of protein study Bradford, 1976). It is worth noting that there is no single most significant protein assay method known to cause utterly perfect results. This, thus, means that it all depends on the desired speed, sensitivity accuracy, the nature of protein, as well as the nature of other components in the protein sample. One such true and accurate method of protein concentration determination involves using the acid hydrolyzing of a portion of sample and carrying out amino acid analysis on the hydrolyzate (Bradford, 1976). However, this method is somewhat expensive and a time consuming. There are various methods of protein quantification such as the Ultra violet Absorbance, Lowry Assay and BCA assay (Mathews, 2000). This experiment employed the Bradford protein assay method in determining of protein. The experiments entail a spectroscopic, analytical procedure used in measuring the concentration of proteins in any solution. The procedure in this method relies on the binding of the dye Coomassie BrilliantBlue G-250 to protein in the sample, in which the dye is proportional to the protein concentration. The bradford dye-binding assay is a colorimetric assay method for measuring protein concentration in solution samples. Much of it involves the binding of Coomassie Brilliant blue to protein. In this case, there is no interference from carbohydrates such as sucrose or from cat ions. However, some of the detergents like sodium dodecyl sulfate, as well as triton x-100 are capable of interfering with the assay, and the alkaline solutions. In this particular experiment, blank alongside six other test tubes were provided with a certain volume of Bovine albumin standard and the volume of some distilled water. The 1.5 mL of the unknown protein was constituted in the fourth test tube. The absorbance was then found through measuring it with the use of the Ultraviolet-visible spectrophotometer. The protein concentration was the computed with the help of the dilution equation. The determination of the unknown protein concentration was achieved through the use of two methods. These were linear regression method and the graphical methods. Introduction Quantitative determinations of proteins is the most accurate yet more rapid method for estimating of protein concentration in the field of protein study. The Quantitative determinations of proteins can be titrimetric-elemental, gravimetric or spectroscopic (Stoscheck, 1990). An assay originally described by Bradford has become the most preferred method for quantifying protein in many laboratories in recent years. This is a spectroscopic technique that is simpler, faster, and more sensitive than the Lowry method of protein estimation (Zor & Selinger, 1996). If it is compared with the Lowry method, the technique is subject to somewhat less interference with common reagents and non-protein components of biological samples. The Bradford assay is based upon the binding of the dye Coomassie Brilliant Blue G-250 into protein, in which case the dye is said to be proportional to the concentration of the protein. For cases where there is no protein, the solution becomes red-brown while in acidic solution and whenever the protein binds, the pKa of the dye doe shift bringing about the change in color of the dye to blue. Studies have shown that the free dye exists in four different ionic forms, in which case the pKa values are 12.4, 1.15, and 1.82. On the contrary, the more anionic blue form of the dye binding to protein contains the absorbance maximum at the wavelength of 590 nm. However, the assay is more sensitive to about values ranging between 20 to 200 μg protein. This implies that the quantity of proteins found using the process of determining the amount of dye in the blue ionic form. It is often achieved through measuring the absorbance of the solution at 595 nm of wavelength. However, there are also common disadvantages of using this method in quantification of protein in a given sample. The Bradford assay method is inhibited by the presence of detergents (Zor, Selinger, 1996). For instance, Sodium dodecyl sulfate, which is a common detergent, is found in protein extracts since it is used to lyse cells by disrupting the membrane lipid bilayer of a cell. While other detergents interfere with this assay at high concentrations, the interference as a result of SDS is of two different modes, and each occurs at a different concentration. When Sodium dodecyl sulfate (SDS) concentrations are below critical micelle concentration, CMC, in the Coomassie dye solution, the detergent binds well with the proteins that are present in the sample. As a result, the protein binding sites are inhibited for the dye reagent. This can result in underestimations of protein concentration in solution assayed. Whenever the SDS concentrations rises above CMC, the detergents do associate strongly with the Coomassie dye green form, thereby, making the equilibrium shift, hence, produce more of the blue form of the dye. Consequently, this causes an increase in the absorbance at 595 nm independent of the presence of protein in the sample (Noble & Bailey, 2009). This method of the protein assay is also linear over a short range, typically from ranging from 0µg/ml to 2000 µg/ml, often making dilutions of a sample necessary before analysis. Principle of the test The test is based upon the observations in which the absorbance maximum for the acidic solution of Coomassie Brilliant Blue G-250 changes from the value of 465 - 595 nm when bound on protein. The hydrophobic, as well as the ionic interactions, do stabilize the anionic form of the dye and causes a color change. The assay is quite useful because the coefficient of extinction of the dye-albumin complex solution is often constant on a 10-fold concentration range. OBJECTIVE In this experiment, key objective was to quantitatively determine protein concentration in a given samples through Bradford assay and to draw the standard curve by plotting the 595nm against the reagent blank. Apparatus and Materials In this lab Experiment, the apparatus and materials used in the assay include the Lyophilized Bovine serum albumin (BSA) standard, Micropipettes, stop watch, distilled water, computer, test tubes, Coomassie Brilliant Blue 1 and a UV- Spectrophotometer. PROCEDURE In this experiment, the commercially available version of the Bradford protein assay (Bio- Rad), were used. The assay consisted two different protocols, a standard one of the linear range 200-1400 ug/ml and a micro-assay protocol of the linear range 1-20ug/ml. In this experiment,the standard protocol was used. Following the procedure, the initial step involved obtaining and labeling the 6 large test tubes from 1s to 6s. Then the next step involved preparing the BSA standard curve dilutions using micropipettes. It was then followed by labeling another set of 9 test tubes. 5 mLs of the diluted dye reagent were then added to each of the test tubes and the standard protein concentrations with the unknown sample solutions mixed with the diluted dye reagent as indicated in table 2 on the manual and the dilution factor of the unknown samples noted. It was then time to do the incubating of all the test tubes at room temperature for 5 minutes. This was followed by lauching of the program by clicking the Data Studio icon on the Desktop making sure the colorimeter had been plugged into the USB interface to help the Sensor bes calibrated with a 5ml distilled water. The step that followed involved transferring the content of the test tubes to the Colorimeter cuvettes and the cuvettes with colorimeter carefully inserted. The absorbance at wavelength 610 nm (orange) was recorded in Data Studio, and the Start key clicked to begin recording. After 10 seconds, the data recording was stopped by clicking the Stop button. The next step that followed was double clicking the Table on the Display menu then double clicking Orange (610nm) Absorbance. This displayed all the absorbance values that had been measured in 10 seconds. The average values were then recorded in the table in Lab 5.1 Experiment Report sheet. The Data Studio file was then saved on my Documents in the Desktop. The next step was identifying each data (rn) in the Summary window to the left of the graph by clicking once on the run, then again to rename. Finally, the protein solutions were disposed off as directed after the run ended, and finally the cuvettes were thoroughly cleaned. Response to Question d. The protein value for the nutritional labels of the unknown samples were as follows The sample A (2% milk) was 2680µg/mL, that of sample B (protein drink) was 9120µg/mL, and that of sample C (soy milk) was 2580µg/mL. From this result, sample C had the least concentration of proteins followed by sample A while sample B had the highest concentration of 9120µg/mL. The experimental data obtained compare truly to the nutritional labels indicated. The protein drink is expected to have more protein concentration levels than both the 2% milk and soy milk and same is expected of 2% milk in comparison to soy milk. This is confirmed from this experimental result as shown above. The prediction of my results did not deviate significantly from the experimental results. I t was expected that the protein drink was to have more protein concentration levels than both the 2% milk and soy milk and same were also expected of 2% milk in comparison to soy milk and thus confirmed by the experimental results obtained. Results The experimental results were tabulated as shown. The Data Table Table 1: Absorbance of BSA standard dilutions Test tube Final standard protein concentration (ug/ml) Absorbance at 610 nm (average in 10 seconds) 1 900 1.863 2 699.84 1.561 3 499.63 1.490 4 399.92 1.318 5 200.16 1.164 6 0 1.827 Absorbance of the unknown samples Unknown sample Absorbance at 610 nm Dilution factor A (2% milk) 1.365 1/40 B (protein drink) 1.318 1/80 C (soy milk) 1.561 1/20 DATA ANALYSIS AND DISCUSSION In this section, data analysis involved responding to a series of questions as follow. Response to Question A. The results of the standard BSA dilutions were plotted as shown in the above table. This shows the standard serial dilution of BSA and dilutions of our unknown in this experiment study. The fractions shown are the dilution factors with 1/40, for example, representing 1 part sample to 39 parts of distilled water for an end sample volume 1/40th that of the total volume. Response to Question B. The above results were used to graph the standard curve. The axes used are labeled as y=A, 610 nm and x=ug/mL). The slope of the standard curve is as indicated on the graph. y= 0.000x + 1.432. R2= 0.075 Figure 1: Colorimetric Assay Chart The above chart shows the results from the spectrophotometer reading of all absorbance of BSA standard dilutions Table 1. Each blue point represents a standard BSA dilution analyzed for the concentration value. From the above blue points, an R2 line was drawn and a linear equation calculated. From the linear equation, the concentrations for the unknown samples that fit between the absorbance values of the standard dilutions were calculated. Response to Question C The protein concentration of the unknown samples can be determined by the use the curve and data from Bradford. For calculation of the Protein Sample Dilutions concentration values the equation derived from the R2 line y= x a + b, where y is the absorbance and x is the concentration is used with the unknown absorbance values substituted for “y” and the equation solved for “x”. From these concentrations of the diluted protein samples, the value was multiplied by the inverse of the dilution factor in to find the original concentration. The concentration of protein in unknown sample A. From the equation y= xa + b, 1.365 =0.001 x + 1.432 0.001x=0.067 X=67 multiplied by its dilution factor of 40=2680µg/mL. The concentration of protein in unknown sample B y= xa + b, 1.318=0.001 x + 1.432 0.001x=0.114 X=114 multiplied by its dilution factor of 80=9120µg/mL. The concentration of protein in unknown sample C y= xa + b, 1.561=0.001 x + 1.432 0.001x=0.129 X=129 multiplied by its dilution factor of 20=2580µg/mL Response to Question d The protein value for the nutritional labels of the unknown samples were as follows The sample A (2% milk) was 2680µg/mL, that of sample B (protein drink) was 9120µg/mL and that of sample C (soy milk) was 2580µg/mL. From this result, sample C had the least concentration of proteins followed by sample A while sample B had the highest concentration of 9120µg/mL. The experimental data obtained compare truly to the nutritional labels indicated. The protein drink is expected to have more protein concentration levels than both the 2% milk and soy milk and same is expected of 2% milk in comparison to soy milk. This is confirmed from this experimental result as shown above. The prediction of my results did not deviate significantly from the experimental results. I t was expected that the protein drink was to have more protein concentration levels than both the 2% milk and soy milk and same was also expected of 2% milk in comparison to soy milk and thus confirmed by the experimental results obtained. Conclusion Through the experiment, the objectives were met by solving and determining the concentration of the unknown protein solutions labeled A, B and C through the application of the linear regression method, as well as plotting the standard curve by absorbance against concentration value of the standard. Using the standard curve, the unknown protein solutions had a concentration of 2680µg/mL, 9120µg/mL and 2580µg/mL respectively. References Bradford, M. (1976) A dye binding assay for protein. Anal. Biochem. 72:248-254. Stockinger Lab. Mathews C. K. et al., (2000) Biochemistry: 477- 111. Addison Wesley Longman, California. Stoscheck, C. M. (1990). Quantitation of Protein. Methods in Enzymology 182: 50-69 Zor, T.; Selinger, Z. (1996), "Linearization of the Bradford protein assay increases its sensitivity: theoretical and experimental studies", Anal. Biochem. 236: 302–308. Noble, J.E.; Bailey, M.J.A. (2009), "Quantitation of Protein", Methods Enzymol. 463: 73–95. Read More