Standard curve Preparation for Quantification of Chicken Egg white albumin using Bradford assay

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Standard curve Preparation for Quantification of Chicken Egg white albumin using Bradford assay

Quantifying protein concentration is a very significant process 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. There are several of methods that can be use for protein quantification. Each has its own advantages and disadvantages. One of which is the Bradford assay, which is commonly used because of its simplicity, wide estimated working range, and sensitivity to molecules of interest. Chicken egg 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 complex with certain amino acids. A standard curve was created from the raw data of absorbance reading. From the Pearson’s Rho correlation, it was shown that the absorbance and concentration has a strong positive relationship. The chemical reactions involve in this assay was also analyzed and understood.

INTRODUCTION

Proteins are very important 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 some 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 sensitive enough to detect even the smallest protein content in order to have an accurate result. The assay should also be specific to the component that is to be quantified. This is to avoid contaminants 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-colorimetric 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 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 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 Folin–Ciocalteu reagent under alakaline 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 it’s 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 instead of Folin–Ciocalteu 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 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 make a standard curve in every assay perfumed.

Pearson’s Rho correlation is used to verify the linear relationship between the two 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 Pearson’s Rho Correlation. It is also very important to understand the processes involved with protein quantification. This experiment also makes one to be extra 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 value will be obtained.

MATERIALS AND METHODS

Before anything else, the UV/VIS Spectrophotometer was turned on before starting 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, 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 known concentrations were transferred to six different microfuge tubes respectively. The concentrations of the reagents were the following:

Table 1: Volume of stock solutions for the preparation of different albumin concentrations

Standard no.

Bradford Reagent (ul)

Phosphate Buffered Saline (ul)

Chicken Egg White Albumin (ul)

Concentration (ug/ul)

Blank

500

500

0

1

500

420

80

0.8

2

500

340

160

1.6

3

500

260

240

2.4

4

500

180

320

3.2

5

500

100

400

4

The 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 placed 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 Pearson’s Rho correlation were made from the raw data of absorbance readings.

RESULTS

Table 2: Series of known concentrations of Albumin

Standard no.

Bradford Reagent (ul)

Phosphate Buffered Saline (ul)

Chicken Egg White Albumin (ul)

Concentration (ug/ul)

Blank

500

500

0

1

500

420

80

0.8

2

500

340

160

1.6

3

500

260

240

2.4

4

500

180

320

3.2

5

500

100

400

4

From 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: Pearson’s Rho Correlation of the Absorbance readings at 595nm and Albumin Concentrations

  1. Trial 1; (b) Trial 2; (c) Mean absorbance of the two trials

A.

Standard no.

Concentration (ug/ul)

Trial 1 (A)

Blank

0.0000

0.0000

1

0.8000

0.7170

2

1.6000

0.6750

3

2.4000

0.3790

4

3.2000

0.7190

5

4.0000

0.8640

Pearson's r Correlation

0.679886127

B.

Standard no.

Concentration (ug/ul)

Trial 2 (A)

Blank

0.0000

0.0000

1

0.8000

0.0510

2

1.6000

0.5480

3

2.4000

0.7740

4

3.2000

0.7990

5

4.0000

0.4250

Pearson's r Correlation

0.710839544

C.

Standard no.

Concentration (ug/ul)

Mean absorbance (A)

Blank

0.0000

0.00

1

0.8000

0.3840

2

1.6000

0.6115

3

2.4000

0.5765

4

3.2000

0.7590

5

4.0000

0.6445

Pearson's r Correlation

0.84783844

The absorbance value of the five albumin concentrations were measured twice. With regards to the Pearson’s Rho Correlation of each, Trial 1 shows that the Albumin concentration and Absorbance at 595nm has a strong 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 Peason’s 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 slope 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 Pearson’s 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 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 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 Pearson’s 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 based on the slope of the line equation.

DISCUSSION

Bradford 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 Pearson’s 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 Pearson’s 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 Pearson’s 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 Pearson’s 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 that’s why 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 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 RECOMMENDATIONS

Bradford 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 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 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 Pearson’s 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 CITED

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Agricola[Internet]. 2006 [cited 2014 January 20];3(3). Available from

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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.

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colorimetricassays, based upon Lowry and Bradfordtechniques, to estimate total protein in soil extracts. Soil Biology & Biochemistry [Internet]. 2013 [cited 2014 January 20];67(100):166-173. Available from: doi:10.1016/j.soilbio.2013.08.017

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