Determination Of Protein Concentration A Variety Of Methods

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There are a variety of methods that can be used to determine protein concentrations. One specific method uses the Bradford assay, a colorimetric protein assay, which involves the binding of Coomassie® Brilliant Blue G-250 to proteins under acidic conditions. Coomassie Brilliant Blue is known to bind to basic and aromatic amino acid residues.1 Upon binding to a protein, the absorbance maximum for the Bradford reagent shifts from 465nm to595nm. The absorbance of the dye at 595nm is indicative of the protein concentration. In order to successfully determine the unknown concentration of the bovine serum albumin (BSA), a standard calibration curve was created via the serial dilution of a standard solution of bovine serum albumin(BSA). The absorbance of the BSA standard was analyzed after the Bradford reagent was mixed with the solutions according to the experimental procedure.2 The calibration curve was determined accordingly and followed the equation y= 0.0018x + 0.3978 Subsequently, dilutions were made of the BSA of unknown concentration according to the standard Bradford assay procedure.2 Following the dilutions, the absorbences of the samples were determined via a spectrophotometer. Consequently, the concentration of the unknown samples was determined by multiplying the values of the absorbences by the dilution factor used. The concentration of the unknown sample of protein was determined to be 2323.3 with a deviation of ± 598. Therefore, the complete expression was 2323.3 ± 598. During the experiment, it was observed that the Bradford Assay was an effective method for determining unknown concentrations of protein samples of about 1µg/mL. This method proved to be a quick and fairly accurate approach for determining an unknown protein concentration because this approach is highly sensitive to low protein concentrations.

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Introduction:

The Bradford protein assay is a spectroscopic analytical method used to measure the concentration of protein samples in solution. It is a popular protein assay because it is fairly simple to conduct and is sensitive to relatively minute concentrations of proteins in sample. The framework of the Bradford assay relies on the reagent Coomassie Brilliant Blue G-250 dye which binds to proteins under acidic conditions. The dye reagent effectively reacts with arginine residues and less effectively with phenylalanine, tryptophan, tyrosine, lysine, and histidine residues. The binding of the dye to a protein under the appropriate conditions causes a shift in the wavelength of maximum absorption from 465nm to 595 nm.1 The concentration of the protein is then directly related to the absorption values of the samples at 595nm.

Most commonly, a calibration curve is constructed using bovine serum albumin (BSA). The Bradford assay requires the reagent Coomassie Brilliant Blue G-250 dissolved in an acidic solution.3

Advantages of the Bradford assay encompass the observance a stable color which varies according to proteins. The dye's response to protein sample is only linear for a small range of the total sample group. An advantage of the reagent, Coomassie Brilliant Blue G-250, is that has a sensitivity of about 30ng.4However, some disadvantages include: the discoloration of protein samples, variation between different protein samples, and the destruction of the protein sample used in the assay. Due to the variation between proteins, the choice of the standard is quite important.5

The experiment conducted aimed to acquire, through the use of the Bradford assay, a standard curve for the bovine serum albumin solutions and to ultimately determine the concentration of an unknown sample of bovine serum albumin. The standard curve is obtained by performing serial dilutions of the standard samples and then adding the Bradford reagent. First, A blank sample in a disposable cuvette is prepared in which water and the Bradford reagent are added. The absorbances of the blank and the protein samples are recorded through the use of s spectrophotometer. A standard curve can then obtained by graphing the absorbance of the samples versus their concentrations. To find the concentration of the unknown samples, dilutions were performed on the scale the of a 10x and 50x dilution factor so that the relative concentrations fall in the range of .01 to 0.1 . Finally, after the absorbances of the unknown samples are determined, the concentration of the unknown is determined by finding the location on the graph that corresponds to the absorbance of the standard. Subsequently, the concentration of the unknown sample can be found by multiplying the concentration found from the graph by the dilution factor.

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Experimental:

The standard and unknown solution of bovine serum albumin along with any chemicals used was obtained from Sigma Aldrich unless noted otherwise. The de-ionized water used in this experiment was obtained from the Baylor Science Building located in Waco, Texas. The spectrophotometer used was a Beckman DU 520 General Purpose Ultraviolet/Visual Spectrophotometer manufactured in Fullerton, California.

In this experiment, serial dilutions were made of a standard solution of BSA using de-ionized water to give six samples of standard solutions ranging in concentration from 117 to 1000 . Each test tube was covered with para-film to prevent evaporation of the sample. Shortly thereafter, a 10x dilution of the unknown sample(A1) was made. This was performed by adding .9 mL of de-ionized water to .1 mL of the unknown BSA sample. Next, a 50x dilution was performed for the sample A2 where .4mL of de-ionized water was added to .1mL of the unknown BSA solution from A1. After this, .1mL of de-ionized water was pipeted into an empty plastic cuvette along with the Bradford reagent. Each of the BSA samples of unknown concentration were pipeted(.1mL) into clean and labeled test tubes. Approximately, 3mL of dye was added to each of the test tubes. The test tubes were shaken gently to ensure a homogenous solution and then allowed to incubate for approximately 7 minutes. The Beckman Spectrophotometer was calibrated to read at a wavelength of 595 nm. The blank cuvette which contained the de-ionized water and the Bradford reagent was run through the spectrophotometer. Next, the absorbences of the standard BSA solutions were recorded by running them through the spectrophotometer starting with the least concentrated and going to the most concentrated. Using the same method mentioned above, the absorbances for the unknown A1 and A2 samples were measured and recorded. The recorded data can be viewed in the accompanying tables that follow.

Results/Error Analysis:

Serial Dilution Table

Table 1

Sample

BSA vol. (mL)

Water vol. added(mL)

Dilution Factor

Overall Dilution Factor

Absorbance

Concentration of BSA (µg/mL)

1

0.7

0.3

7/10.

1/2.

1.458

1000

2

0.7

0.3

7/10.

7/20.

1.421

700

3

0.7

0.3

7/10.

49/200.

1.253

490

4

0.7

0.3

7/10.

343/2x10^4

1.081

343

5

0.7

0.3

7/10.

2401/2x10^6

0.831

240.1

6

0.7

0.3

7/10.

16807/2x10^8

0.676

168.1

7

0.7

0.3

7/10.

117649/2x10^10

1.118A

117.6

AError for this value, will be discussed in the error section of this report.

* From 2000 µg/ml BSA stock solution in water.

Unknown Dilution Table.

Table 2

Sample

Dilution factor

Absorbance

Blank

/

0

A1

1/10.

0.5338

A2

1/50.

0.1082

Unknown samples: Class data

Table 3

A1 Samples from class

A2 Samples from class

1) 0.424

0.062

2) 0.490

0.074

3) 0.604

0.139

4) 0.512

0.101

5) 0.791

0.138

6) 0.382

0.135

Attached you will find a copy of the raw experimental data that

accompanies the data in table 1 & 2

Graph 1: Absorbance vs Concentration of Standard BSA

Results/Error Analysis Continued.

In order to determine the unknown concentration of the BSA sample, the standard curve had to be analyzed. The equation of the line of best fit for the points above was found to be : Y= .0018x + .03978. It is important to note that three points were omitted from the graph(above) due to their non-linear relationship with the rest of the data points. Had these points been included in the graph the equation of the line would have been Y= 0.001x + .3559 and the R2 value would have been 0.8586. Thus, when calculating the unknown concentrations, the values that would have been obtained from this equation would be negative. As the date points began to taper off, it was a sign that the linear nature of the line was disappearing. The R2 value of the line was 0.972. This indicated that the points fell very close to the line of best fit. The unknown concentrations were determined by finding the concentration that corresponded to the absorbance. This was then multiplied by the dilution factor to give the actual concentration of the unknown BSA solution samples. The mean value for the unknown concentration was determined to be is 2323.3 ± 598 .

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Finally, it is important to point out that in Table 1, the absorbance value for sample #7 was found to be 1.118. This absorbance value was omitted from the standard curve because it did not follow the linear relationship of the standard dilution curve. Furthermore, it was determined that this abnormal value was a result of not taking 100 µL out of tube 7 and then adding the Bradford dye. Rather, what occurred was the final volume that accumulated in tube 7 was not taken account for and thus resulted in an abnormally high absorbance value. A potential source of error that could have affected the value of our absorbance values could have possibly come from experimental error. This could have come in the form of our spectrophotometer not operating properly due to improper calibration or the UV lamp malfunctioning. Consequently, this would have resulted in a deviation of absorbance values and affected the concentration values of the protein samples of unknown concentration.

Calculations:

To perform the calculations below, the equation of the standard curve was used alongside graphical analysis.

A1: Y= .0018x + .03978

.534=.0018x + .03978

x= 274.6µg/mL

Because this was a 10x dilution, the original concentration is:

(274.6µg/mL)(10)= 2746 µg/mL

A2: Y= .0018x + .03978

.1082=.0018x + .03978

x= 38.01µg/mL

Because this was a 50x dilution, the original concentration is:

(38.01µg/mL)(50)= 1900.6 µg/mL

Mean Value: =

= 2323.3

Standard Deviation Calculation:

= 597.8

This value is the standard deviation for the unknown concentrations.

Therefore, the deviation is 2323.3 ± 598 .

Mean calculation for unknown class data

A1 = .534

A2 = .1082

Discussion:

The objectives of this experiment were met. Using the Bradford method, it was possible to construct a standard curve for the standard solution of bovine serum albumin and determine the unknown concentration of a bovine serum albumin solution. By using the standard curve constructed from the standard solutions, it was possible to determine the concentrations of the unknown dilutions through the use of the Bradford method.

Usually, it is observed that when the concentration of a protein sample increases, the absorbance also increases. This occurs because the specific wavelength of light emitted is absorbed by the delocalized pi electrons in the aromatic side chains of the amino acids tyrosine, tryptophan, and phenylalanine. Also, Beer's law states that the absorbance is equal to the path length times the concentration times the coefficient.  Therefore, it is the increase in concentration of aromatic side chains that causes the absorbance to increase as the amount of protein is increased. With the addition of the Bradford reagent ( Coomassie Brilliant Blue G-250) and successful binding to proteins, the wavelength of maximum absorption of the dye shifts from 465nm to 595nm. According to the data for the standard curve, it was observed that there was a positive linear relationship for the lower protein concentrations. It was also observed that some of the higher values began to taper off and fit a non-linear relationship. Therefore, these "outlier" data points were discarded. The line of best fit for the data points was found to have the equation Y= .0018x + .03978 and an R2 value of 0.9718. The R2 value of 0.9718 indicated that the data points fell within minimal deviation from the line of best fit. Furthermore, the y value in the equation indicated the absorbance while the x value indicated the concentration of the protein.

The unknown concentrations were determined by using the line of best fit. Essentially, the unknown concentration was found by finding the corresponding concentration for each of the measured absorbances. Then, the actual concentrations of the unknown sample of bovine serum albumin could be found by multiplying those concentration values by the dilution factor. It was determined that the average protein concentration of our unknown sample of bovine serum albumin was 2323.3 ± 598 This value was found by averaging the values of the two unknown samples listed in the Calculations section.

Lastly, another route to determining the concentration of a protein sample is through the use of the extinction coefficient of protein. This methodology employs the use of the equation A=ECL. Where A is the absorbance, E is the extinction coefficient value for a certain protein, C is the concentration of the protein in mg/mL, and L is the length of the cuvette used. This method is a more concrete way of determining the concentration of a protein sample and allows one to insert the unknown values and solve for a variable. One drawback to this method is that it does not allow one to interpret the values to a great degree. However, by using the Bradford assay one can construct a standard curve based on the methods described in the paper and interpret the results in a more graphical manner. This method relies on plotting the standard curve and determining the unknown protein concentrations by using points on the line of best fit that coincide with the absorbances recorded for the samples of unknown concentration.