Quantification of Chicken Egg White Albumin

Quantification of yellow(a) Egg White AlbuminStandard curve Preparation for Quantification of Chicken Egg clear albumin using Bradford chequeQuantifying protein submerging is a very significant impact for analyzing protein. It is essential in order to identify, characterize, and purify proteins, and this can also be use in medical researches by aiding in diagnosis of certain diseases. thither are several of methods that can be use for protein quantification. Each has its own advantages and disadvantages. adept of which is the Bradford essay, which is parking arealy used because of its simplicity, great estimated working range, and sensitivity to molecules of interest. Chicken addict white albumin was used as a protein source. Different concentrations of albumin were prepared and their respective absorbances at 595nm were obtained. This assay uses Coomassie Blue G-250 dye that forms a interwoven with certain amino acids. A stock curve was created from the raw data of absor bance reading. From the Pearsons Rho coefficient of correlativity, it was shown that the absorbance and concentration has a fond verifying relationship. The chemical reactions accept in this assay was also analyzed and understood.INTRODUCTIONProteins are very important in an organism for growth and preservation. forrader 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 many illnesses affect the level of proteins. There are different methods in order to quantify protein concentration. It is important for an assay to be able to be applied to a wide range of concentrations. It would also be good for the assay to be tippy enough to detect even the smallest protein content in order to gift an accurate result. The assay should also be specific to the component that is to be quantified. This is to avoid contaminant s to be detected, such as cell components, macromolecules like carbohydrates, nucleic acid and lipids. There are different methods on quantifying proteins. One of which is the Non- annotateimetric procedures. This includes determination of nitrogen derived from proteins, analysis of amino acids, and find out of dry matter material of protein. The one that is common in this modern time 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, cupric (Cu2+) ions are creation 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 high concentration of proteins since it is quite insensitive (Dennison, 2003).Lowry assay starts with a protein-copper complex just like the product in the Biuret assay. In Lowry assay it is then followed by the reduction of FolinCiocalteu reag ent under alaka grade 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 non 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 instead 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 chiefly 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 essay 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 demand before doing any 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 rile a standard curve in every assay perfumed.Pearsons Rho correlation is used to verify the linear relationship betwixt the cardinal 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 apprehend the processes involved with protein quantification. This experiment also makes one to be extra carefu l with the research science lab procedures so that accurate data can be obtained.In a higher concentration, there are more particles involved, and so when a UV ethereal strikes, more particles will be able to absorb it and higher absorbance value will be obtained.MATERIALS AND METHODSBefore anything else, the UV/VIS Spectrophotometer was turned on before kickoff the procedures in order for the machine to warm up and function properly this avoids interfering with the data reading. Enough amounts of Chicken egg white albumin 10mg/mL, inorganic 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 cognise concentrations were transferred to six different microfuge tubes respectively. The co ncentrations of the reagents were the following bow 1 Volume of stock solutions for the preparation of different albumin concentrationsStandard no.Bradford Reagent (ul)Phosphate Buffered Saline (ul)Chicken Egg White Albumin (ul)Concentration (ug/ul)Blank viosterol five hundred01500420800.825003401601.635002602402.445001803203.255001004004The 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 side panel. The cuvettes were gently move in the spectreophotometer. The absorbance of each concentration was read at 595 nm. The procedures were done for two 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 AlbuminStandard no.Bradford Reagent (ul)Phosphate Bu ffered 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 Correlation of the Absorbance readings at 595nm and Albumin Concentrations struggle 1 (b) Trial 2 (c) Mean 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 Correlation0.679886127B.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 (ug/ul)Mean absorbance (A)Blank0.00000.0010.80000.384021.60000.611532.40000.576543.20000.759054.00000.6445Pearsons r Correlation0.84783844The absorbance value 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 blind drunk positive relationship. While trial 2 and the mean 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 amongst 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 slope has a positive value giving an upward command 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 stro ng 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. But for standard no.5, the absorbance of 4.00 ug/ul Albumin went heap 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 mean 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 0.84783844. 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 b ased 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 closer 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 stan dard 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 must 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.One 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 spectrophotometer. The particles already absorbed an amount of light thats why the light they absorbed in the spectrophotometer became less than expected. Temperature can also affect the Bradford assay. Since th e 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 touch on the data. Lowering the temperature can increase the absorbance and vice versa (Steinke Shepherd, 1992). The basic and aromatic amino acids are the interest of the Bradford assay (Dennison, 2003). Some 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 since its easy to perform and at the same time good data were gathered. However, it would be discontinue if at least two different assays were performed in order to verify the standard curve for the chicken egg white albumi n and to further understand how the other assay works. Ensuring the consistent temperature in the working place can improve 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. Albumen protein and functional properties of gelation and foaming. ScienceAgricolaInternet. 2006 cited 2014 January 203(3). forthcoming 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. Available from http//books .google.com.ph/books?id=SuXi2WtHnwQCdq=lowry+assaysource=gbs_navlinks_s. Also available in paper copy from the publisher.Doss, D., Sumrall III, W., McElreath D., Jones D. 2013. Economic 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 polyethylene 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. essay 1, Standardcurve preparation for determining protein content.URCO.Redmile-Gordon, M., Armenise, E., White, R., Hirsch, P., Goulding, K. A comparison of twocolo rimetricassays, 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. Fundamental 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 publ isher.