Fathima S. Hussain Cell and Molecular Lab Ms.Dang 2nd April 2018 The method for calculating the unknown concentration of the protein given by using the standard curve that is generated from the given known standard proteins. Abstract Measuring the total concentration of an unknown wheat germ protein in a solution through the Bradford method. The Bradford protein assay is a method for measuring the total protein concentration using the Coomassie brilliant blue G-250 as it joins the total protein in the given sample. The Bradford assay method is one of the most likely methods since it is not time-consuming and less likely to have chemical interferences when compared to other protein assay methods. This method was introduced in the nineteenth century in which it has given numerous amount of accurate results and it’s reproducible.
Within this lab, beers law was implemented to find the concentration of the protein since the spectrometer measures the absorbance within the given solution of the protein and the dye. The protein-dye allows the light to absorb at specific wavelength because the protein-dye complex has the maximum absorbance wavelength at 595nm.The absorption is directly proportional to the concentration of the unknown protein sample given and it is measurable with the usage of the spectrophotometer. To determine the total protein concentration a standard curve has been produced to give the absorption of several solutions with a known concentration of the protein range of 10-100ug. Introduction Proteins are huge macromolecules which are important to all living organisms. In this experiment catalysts and regulation of many cellular metabolic processes are examples of their many functions. A spectrometer has four major components which include light sources, a monochromator, a sample holder and two photodetectors.
All these combined help produce the measurement of light absorbance. A protein concentration cannot as simply be directly measured by light. One way, although not guaranteed for all proteins is, having a large number of aromatic amino acids, which its concentration can be measured by UV absorbance. The usage of UV absorbance will give a good estimate of the total protein concentration with the help of beer’s law since the unknown protein contains no other components(Noble). The Coomassie brilliant blue protein-dye was a method that has been used since the nineteenth century in which it has presented itself with numerous amount of accurate results.
A more confirmed way would be to create colored complex between a protein and a dye. At specific wavelengths, this complex will absorb light which is that in balance to the concentration of the protein. The standard curve is used as a guide for measuring the amount of a substance in a solution of unknown concentration. The substance is a protein in this case. Serial diluting is a useful procedure when measuring concentrations of the sample where it is unknown because it is simpler in the process of making and having a well of range.
Using the serial dilution increase the accuracy within this lab and improves the condition of the assay due to the 10-fold method allowing it for a better result with the Bovine Serum Albumin (Ernst & Zor). The hypothesis of this lab would include a linear relationship amongst the protein and the absorbance because each of the protein is diluted in which it allows the light absorbance to measure more efficient giving a positive linear line. In this lab, the concentration of protein in a solution is being measured using the equation A=elc. This equation is beer’s law which is used to find the absorbance in which c is the concentration of the protein and E stands for the extinction coefficient for each protein, and I is the measurement of the degree of light absorbance is proportional to the amount of to the amount of protein in the solution. The absorbance is set to 594 because the protein binds at that particular wavelength giving an accurate reading of the dilutions(Noble). In this lab, we use a standard curve to determine the concentration of the unknown protein.
The curve standard curve is used to show the relationship between the absorbance and the amount of standard protein in solution will be linear since the free dye concentration produces a linear relationship since the protein-dye binding goes through equilibrium which consists to be more dependent on the concentration of the unknown protein (Ernst; Zor). We can compare the absorbance of the unknown solution to the curve to find the amount of unknown protein in solution. Materials and Methods The materials that were used in the process to determine the total protein concentration of the given sample is the spectrometer in which beers law was applied to measure the absorbance, the cuvettes were used to measure the absorbance with the serial dilutions with the help of pipets, Coomassie brilliant blue protein-dye to help with the measure of serial dilutions, and the unknown protein sample that was given. To begin with, a BSA solution was obtained which is 1.0mg/L and it was labeled from B1-25 for accuracy throughout the lab. With the BSA solution given a serial amount of solution according to the table 1 was mixed thoroughly. The spectrometer was set to a wavelength of A595 in which each tube was used to check the wavelength from the serial dilution.
Using the data collected, a graph was constructed (fig1) to find the standard curve through the given linear equation presented. Next, obtain100ul of the unknown protein solution was obtained and do serial dilution solution by the 10-fold dilution method mentioned in the lab manual. The final volume of each of the tube will have to be 500uL.Afterward, an addition of 50ul of protein solution to 450uL of the buffer to make the dilution and mix it thoroughly with the pipet. Then from each of the diluted solution, take 50uL of the solution and add it to 2.5 mL of Bradford reagent and mix thoroughly. Towards the end, wait for 5 mins and measure the absorbance with the wavelength set to 595nm.
With the absorbance recorded after numerous amount of serial dilution, a graph will be produced to determine the standard curve. With the use of the linear line produced calculate the unknown protein concentration of the given amount. Results Table 1 BSA Standard Curve Data This table mentions the serial dilutions made throughout the experiment and it shows an relationship amongst the absorbance and the BSA. As there is an increase in the BSA through serial dilution there is an increase in the absorbance since the number of NaCl added decrease giving a more diluted solution and light to be absorbed at ease. Table 2 Wheat germ Bradford assay Data This table represents which tube was used to determine the unknown concentration of the unknown solution in which WG2 was used to give the total protein concentration as 2.594mg/mL.
Fig 1. With an increase in absorbance values there’s an increase in protein (ug/tube) leading to an increase in concentration of the protein in solution in which it shows a positive correlation. Standard curve of BSA protein contains 0-50 ug BSA in which it was fitted with a linear equation. TOTAL PROTEIN: y=0.0209*ug(protein)+0.0149 A595=0.0209*ug(protein)+0.0149 A595=0.286 ug(protein)= (0.286-0.0149)/0.0209 =12.97ug Total Protein = 12.97ug/0.05mL = 259.4ug/mL Dilution factor= 10 259.4ug*10= 2594ug/mL = 2.594mg/mL Discussions For this lab, we had to find the protein concentration using the Bradford method and serial dilution. We started out by finding the standard cure of our given BSA (solution whose concentration is unknown: 1mg/ml). We did so by adding to 6 different sample tubes, the amount of BSA, 0.15M NaCl and Bradford reagent that was given by the manual.
Afterward, we used the spectrometer to find the absorbance levels of the solution. In this lab, we measured the concentration through spectrophotometry method in which the beer law mentioned that the absorbance of the unknown protein is proportional to the protein absorbance. In the Bradford assay method, the Coomassie brilliant blue G250 which binds to the blue color of the protein. The dye-protein absorbs accurately at 595 as the wavelength. Beer’s Law is represented in the formula A=elc in which the absorbance is directly proportional to the concentrated protein solution. The spectrophotometer is used in the Bradford method to determine the protein concentration in which it measures the light absorbance in the cuvette’s that were used throughout this experiment.
After the data has been collected, a standard curve was produced with the serial solution of BSA since it has a known concentration. The standard curve was used as a basic foundation to measure the concentration of the protein in the unknown solution. The BSA- dye solution was used throughout the experiment to determine the unknown concentration of the given sample. The curve that was produced gave a linear line which was expected from the beginning since as the dilution of the protein increased the absorbance also increased since the light absorbed since as the samples were diluted throughout to find more of an accurate recording to determine the calculation of the unknown protein.Using this data, we made a graph in which we derived the proteins as the x-axis and the absorbance as the y-axis. From this, we concluded that we did somewhat of accurate numbers since the values of the absorbance lied on the linear line that was constructed with the data collected. After this, we had to find the concentration of the protein in our unknown original solution.
We did this by serial dilution and then finding the absorbance levels of this solution. We chose the value that fell in the middle region of our standard curve because the middle range can be determined accurately which was the WG2 tube with the A595 of 0.2329(fig 1). The most effective method to determine the concentration of the unknown protein is to use serial dilution since it will give us a value within the middle range of the standard curve. The serial dilution was efficient since it allowed us to be productive in a short period of time and sampled different dilutions to give us more accurate readings.
We used this value and set it to y in our linear equation of the standard curve line, and found (0.0209)x, which is the amount of protein (ug/tube).We then divided this amount by 0.05 mL(the volume of standard tube and unknowns) and multiplied it by 10(dilution factor of W2) and got 2549 ug/ml. Finally, we converted this amount to 2.549 mg/ml which is the protein concentration of our original unknown assigned. In this lab, Bradford protein assay was used to determine the protein concentration of the unknown wheat germ extract that was given. BSA was used for the determination of the standard curve.
The determined protein concentration of the wheat germ extract is 2.549 mg/mL. Literature Cited Noble, J. E. (2014).
Chapter Two – Quantification of Protein Concentration Using UV Absorbance and Coomassie Dyes. Quantification of Protein Concentration Using UV Absorbance and Coomassie Dyes,17-26. doi:10.1007/978-1-61779-821-4_3 Zor, T., & Ernst, O. (2012). US National Library of Medicine National Institutes of Health. Linearization of the Bradford Protein Assay. doi:10.3897/bdj.4.e7720.figure2f
