Quantification of Chicken Egg White Albumin

Quantification of sensationalistic nut White white example wind Preparation for Quantification of squawker Egg unclouded albumin using Bradford chequeQuantifying protein immersion is a genuinely signifi backt run for analyzing protein. It is essential in localise to identify, characterize, and purify proteins, and this provide in deal manner be use in medical researches by aiding in diag noneis of certain diseases. There ar several of methods that can be use for protein quantification. Each has its own advantages and disadvantages. wizard of which is the Bradford halt, which is commonly used because of its simplicity, wide estimated working range, and sensitivity to molecules of interest. Chicken globe snow-clad albumin was used as a protein source. Different concentrations of albumin were prep atomic number 18d and their respective absorbances at 595nm were obtained. This assay uses Coomassie Blue G-250 soil that forms a tortuous with certain amino stiflings. A measuring fold was created from the raw(prenominal) data of absorbance reading. From the Pearsons Rho correlational statisticsal statistics, it was shown that the absorbance and concentration has a firm corroborative kindred. The chemical reactions accept in this assay was also analyzed and understood.INTRODUCTIONProteins argon very outstanding in an organism for growth and preservation. Before analyzing a protein, it requires to determine the quantity of proteins present. The result of this will be useful in characterization and purification of proteins, in identification and in diagnosis of diseases, since round illnesses affect the level of proteins. There are different methods in order to quantify protein concentration. It is important for an assay to be satisfactory to be utilise to a wide range of concentrations. It would also be good for the assay to be in the altogether enough to detect even the smallest protein content in order to have an immaculate result . The assay should also be specialized to the component that is to be quantified. This is to avoid contaminants to be detected, such as cellphone components, macromolecules like carbohydrates, nucleic acid and lipids. There are different methods on quantifying proteins. ace of which is the Non-colorimetric procedures. This includes determination of nitrogen derived from proteins, analysis of amino acids, and find out of ironic matter material of protein. The one that is common in this modern cartridge clip is the Colorimetric methods of quantitation. This is because of the technological advancements available like usage of spectophotometers (Ninfa et al., 2009)In biuret assay, cuprous (Cu2+) ions are being reduced to cuprous (Cu1+) ions by the proteins. This cuprous ions forms a complex with the peptide bonds yielding a blue colored complex. This assay requires spunky concentration of proteins since it is quite in slight (Dennison, 2003).Lowry assay starts with a protein-copp er complex in force(p) like the product in the Biuret assay. In Lowry assay it is then followed by the reduction of FolinCiocalteu reagent under alaka task conditions. Cuprous ions are the ones involve in the process of reduction, resulting to a intense blue color. Lowry assay is more sensitive unlike the biuret assay however, since its very sensitive, it can detect other components that are not of interest just like detergents (Dennison, 2003).BCA Assay is the same as the Lowry assay, but bichoninic acid (BCA) is the one being reduced by the protein kinda of FolinCiocalteu reagent. BCA assay is sensitive but not to other contaminants. It is more sensitive to carbohydrates, lipids and other substances (Dennison, 2003).Bradford assay, which is the most commonly used colorimetric method, uses Coomassie Blue G-250 dye. This dye forms a noncovalent bond with proteins primarily basic amino acids (arginine, lysine and histidine). This complex results to a blue form in color. This assay is sensitive, accurate and can be done quickly (Redmile-Gordon et al., 2012).The source of protein for this experiment is the chicken egg white albumin. Studies consisting of iron-chelation, protease inhibition, immunoregulation, etc. uses chicken egg white albumin as well. Purification of the albumin is very much needed onwards doing each experimental procedure with the protein (Geng et al., 2012). In fact the albumin is consisting of 385 amino acid residue (Alleoni, 2006).In determining the protein concentration, it is very important to make a standard curve in every assay perfumed.Pearsons Rho correlation is used to verify the run alongar relationship between the devil variables involve in this experiment, absorbance and albumin concentration (Statstutor, n.d.).This study aims to verify how the concentration of proteins can affect the absorbance of the sample. It is also to make a standard curve for Chicken egg albumin and to know the Pearsons Rho coefficient of correlation . It is also very important to consider the processes involved with protein quantification. This experiment also makes one to be scanty careful with the laboratory procedures so that accurate data can be obtained.In a higher concentration, there are more particles involved, and so when a UV light strikes, more particles will be able to absorb it and higher absorbance hold dear will be obtained.MATERIALS AND METHODSBefore anything else, the UV/VIS Spectrophotometer was turned on before starting the procedures in order for the mould to warm up and function properly this avoids interfering with the data reading. rich amounts of Chicken egg white albumin 10mg/mL, Phosphate Buffered Saline (PBS) pH 7.4, and Bradford reagent were obtained in a beaker from the reagent bottle. The beaker which contained the Bradford reagent was covered with paper since the the reagent is very light sensitive and might affect the data. With the use of micropipettors, albumin, phosphate buffered solution and Bradford reagent with cognize concentrations were transferred to half dozen different microfuge tubes respectively. The concentrations of the reagents were the followingTable 1 Volume of stock solutions for the dressing of different albumin concentrations ensample no.Bradford Reagent (ul)Phosphate Buffered Saline (ul)Chicken Egg White Albumin (ul)Concentration (ug/ul) boob euchre d01500420800.82500340clx1.635002602402.445001803203.25500 ascorbic acid4004The solutions were vortex one at a time for 10 seconds. It settled for 10 minutes. After that, the solutions were again vortex for 5 seconds. Then the solutions from the microfuge tubes were transferred to the cuvettes individually. The cuvette was not touched on the flat cheek panel. The cuvettes were gently placed in the spectreophotometer. The absorbance of each concentration was read at 595 nm. The procedures were done for twain trials.The standard curve and determination of Pearsons Rho correlation were made from the raw data of absorbance readings.RESULTSTable 2 Series of known concentrations of Albumin streamer no.Bradford Reagent (ul)Phosphate Buffered Saline (ul)Chicken Egg White Albumin (ul)Concentration (ug/ul)Blank50050001500420800.825003401601.635002602402.445001803203.255001004004From the volume of the stock solutions, different concentrations of albumin were made. The albumin concentration of the standards ranges from 0.0- 4.0 ug/ul and have an increment of 0.8.Table 2 Pearsons Rho coefficient of correlation of the Absorbance readings at 595nm and Albumin ConcentrationsTrial 1 (b) Trial 2 (c) nasty absorbance of the two trialsA.Standard no.Concentration (ug/ul)Trial 1 (A)Blank0.00000.000010.80000.717021.60000.675032.40000.379043.20000.719054.00000.8640Pearsons r correlation coefficient0.679886127B.Standard no.Concentration (ug/ul)Trial 2 (A)Blank0.00000.000010.80000.051021.60000.548032.40000.774043.20000.799054.00000.4250Pearsons r Correlation0.710839544C.Standard no.Concentration (u g/ul)Mean absorbance (A)Blank0.00000.0010.80000.384021.60000.611532.40000.576543.20000.759054.00000.6445Pearsons r Correlation0.84783844The absorbance set of the five albumin concentrations were measured twice. With regards to the Pearsons Rho Correlation of each, Trial 1 shows that the Albumin concentration and Absorbance at 595nm has a healthy electro supportive relationship. While trial 2 and the blotto of the two trials show that the two variables exhibit a very strong positive relationship.Figure 1 Trial 1 Absorbance at 595nm vs. Concentration of Albumin (ug/ul)In trial 1, the Peasons Rho Correlation value is 0.679886127 showing a strong positive relationship between the two variables. From the absorbance value of the standard no.1, there is a decrease in the absorbance in standard no.2 and standard. No.3. Then a sudden increase in absorbance in standard no.4 and standard no.5. And also from the line equation, the gradient has a positive value giving an upward direction and a direct correlation between absorbance and concentration of the albumin.Figure 2 Trial 2 Absorbance at 595nm vs. Concentration of Albumin (ug/ul)Trial 2 shows a very strong positive correlation between the two variables since the value of the Pearsons Rho Correlation value is 0.710839544. Standard no.1 until Standard no.4 shows that the absorbance at 595nm is increasing with the albumin concentration. further for standard no.5, the absorbance of 4.00 ug/ul Albumin went down very quickly. Based on the line equation of the best fit line of this graph, the slope has a positive value giving an upward direction and a direct correlation between absorbance and concentration of the albumin.Figure 3 Mean Absorbance of the two trials at 595nm vs. Concentration of Albumin (ug/ul)The pie-eyed values of the absorbance at 595nm for the two trials were obtained and graph. The mean absorbance and albumin concentration shows a strong positive correlation, having a Pearsons Rho correlation of 0.8 4783844. The graph shows that there are two outliers, standard no. 3 and standard no.4. A direct correlation between mean absorbance and concentration of the albumin is also shown establish on the slope of the line equation.DISCUSSIONBradford assay utilize Coomassie Blue G-250 dye that forms a complex with the basic amino acids and thus having a blue form in color as the outcome (Redmile-Gordon et al., 2012).The concentration of the chicken egg white albumin ranges from 0.0 ug/ul, which is the blank , up to 4.0 ug/ul. This shows an increasing concentration of the albumin, and to have an accurate data, there is 0.8 so that the gap between the concentrations is equal. With this, the absorbance at 595nm can be compared to see the relationship between the two variables.In Pearsons Rho Correlation, the walk-to(prenominal) the value to 1 or -1, the strong is the linear correlation for the two variables (Statstutor, n.d). In trial 1, the Pearsons Rho Correlation value is 0.679886127, and since the value ranges from +.40 to +.69, the Albumin concentration and absorbance value has a strong positive. The graph shows that the absorbance readings are not consistent. Standard no.2 and standard no.3 shows a sudden decrease.The Pearsons Rho Correlation value for trial 2 is 0.710839544. This shows that the relationship between the two variables is a strong positive relationship. This is because for a correlation to be strongly positive, the value essential be +.70 or higher. For this trial, standard no.5 is the outlier. For the mean of the two trials, it could be seen that Standard no.3 and Standard no.5 are the outliers (Fig 3), although there is a strong positive relationship between the two variables based on its Pearsons Rho Correlation value, 0.84783844. ace reason for having a result with outliers is that the solutions containing chicken egg white albumin, phosphate saline buffer and Bradford reagent were already exposed to light even before it was placed in the spect rophotometer. The particles already absorbed an amount of light thats wherefore the light they absorbed in the spectrophotometer became less than expected. Temperature can also affect the Bradford assay. Since the temperature of the environment of the solution may vary throughout the experiment, like the temperature of the hands holding the cuvettes, the place where the solutions were settled, inside the cabinet, it might experience a change in temperature that affected the data. Lowering the temperature can increase the absorbance and frailness versa (Steinke Shepherd, 1992). The basic and aromatic amino acids are the interest of the Bradford assay (Dennison, 2003). virtually other amino acids present in the protein may not be detected by the assay. Different assay for protein quantitation have own advantages and disadvantages that why there is no a standard assay.CONCLUSIONS AND RECOMMENDATIONSBradford assay for standard curve preparation is very suitable for this experiment s ince its easy to perform and at the same time good data were gathered. However, it would be better if at least two different assays were performed in order to verify the standard curve for the chicken egg white albumin and to further understand how the other assay works. Ensuring the consistent temperature in the working place can correct the outliers in the absorbance reading. And lastly being extra careful on the solutions being not exposed to light and any contaminants may improve the data. To conclude, this experiment verifies the positive relationship between absorbance and correlation base on the Pearsons r correlation, since positive value denotes positive linear correlation.The slope from the linear equation for the two trials and the mean also shows a positive one indicating a correlation between the two variables involve.LITERATURE CITEDAlleoni, A. flannel protein and functional properties of gelation and foaming. ScienceAgricolaInternet. 2006 cited 2014 January 203(3). purchasable fromhttp//dx.doi.org/10.1590/S0103-90162006000300013Dennison, C. 2003. A guide to protein isolation Internet. The Netherlands Kluweracademic Publishers 2003 cited 2014 January 20. useable from http//books.google.com.ph/books?id=SuXi2WtHnwQCdq=lowry+assaysource=gbs_navlinks_s. in any case available in paper copy from the publisher.Doss, D., Sumrall III, W., McElreath D., Jones D. 2013. sparing and financial analysis forcriminaljustice organizations Internet. Florida CRC Press 2013 cited 2014 January 20. Available from http//faculty.quinnipiac.edu/libarts/polsci/statistics.html. Also available in paper copy from the publisher.Geng, F., Huang, Q., Wu, X., Ren, G., Shan, Y., Jin, G., Ma, M. Co-purification of chicken egg white proteins using polythene glycol precipitation and anion-exchange chromatography. Separation and Purification Technology Internet. 2012. cited 2014 January 209675-80. Available from http//dx.doi.org/10.1016/j.seppur.2012.05.021Oyong, G. 2012. Basic and advanced techniques in cell and molecular biology. Experiment 1, Standardcurve preparation for determining protein content.URCO.Redmile-Gordon, M., Armenise, E., White, R., Hirsch, P., Goulding, K. A comparison of twocolorimetricassays, based upon Lowry and Bradfordtechniques, to estimate total protein in soil extracts. Soil Biology Biochemistry Internet. 2013 cited 2014 January 2067(100)166-173. Available from doi10.1016/j.soilbio.2013.08.017Statstutor Internet. Pearsons correlation. cited 2014 January 20. Available fromhttp//www.statstutor.ac.uk/resources/uploaded/pearsons.pdfSteinke J., Shepherd A. Effects of temperature on optical absorbance spectra of oxy-, carboxy-, and deoxyhemoglobin. Clinical Chemistry Internet. 1992 cited 2014 January 2038(7)1360-1364. Available from http//www.clinchem.org/content/38/7/1360.longNinfa, A., Ballou, D., Benore, M. 2009. native laboratory approaches for biochemistry andbiotechnology Internet. New Jersey Wiley 2009 cited 2014 January 20.Available fromhttp//books.google.com.ph/books?id=k6_XQwAACAAJdq=fundamental+lab ratory+appoaches+for+biochemistry+and+biotechnologyhl=ensa=Xei=ajbdU uxo2tB7jTgEAved=0CC4Q6AEwAA. Also available in paper copy from the publisher.