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Protein Analysis

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					                       Determination of Protein Content

This lab introduced students how to determine protein content based on % Nitrogen versus
 protein binding. Students also will be introduced to the Dumas and Bradford Dye Binding
                         techniques to determine protein content.

                                    Virginia Smith
                                     983976633

                                 Lab Partner: Jeff Martz

                                   Due Date: 3-15-11
Introduction

The objective of this lab is to introduce the students to determine protein based on %

Nitrogen versus protein binding. Also, the protein content can be determined using the

Dumas and Bradford Dye Binding techniques. The protein content of a sample is important

in proximate analysis in order to determine nutritional content, functional and legal reasons.



Materials and Methods

This experiment was conducted as described in the laboratory manual (Roberts 2011) with

the following minor modifications. Firstly, each student will fill the aluminum foil with 200

mg of nonfat dry milk to be analyzed in Dr. Coupland’s Lab for the Dumas method. Secondly,

the concentrations of BSA of the protein standards were altered.


Results

               0.7

               0.6

               0.5
  Absorbance




               0.4
                                                                  y = 1.0157x - 0.0205
                                                                       R² = 0.9945
               0.3

               0.2

               0.1

                0
                     0   0.1   0.2      0.3         0.4     0.5          0.6        0.7
                                 Concentration of BSA (mg/mL)



Figure 1. Standard curve of the nonfat dry milk sample blanks.
Table 1. Nitrogen analysis of the NFDM samples using Bradford Reagent.
                          Absorb.                       Conc.
         1                 0.3098                      0.3252
         2                 0.3372                      0.3522
         3                 0.3565                      0.3712
      mean                 0.3345                      0.3495
       std                 0.0234                      0.0231
      %CV                  0.0702                      6.6103
 % relative error                                     -1.5493
  % abs. error                                         1.4511


Table 2. Nitrogen analysis of the NFDM samples using Dumas Combustion method.
                          NFDM mass           Nitrogen %         Protein %
         1                  0.1993              5.2581             33.547
         2                  0.2054              5.3165             33.919
         3                  0.2054              5.3006             33.818
         4                  0.2040              5.3945             34.417
       mean                 0.2035              5.3174             33.925
standard deviation          0.0029              0.0570             0.3635
       %CV                  0.0142              0.0107             1.0715
  % relative error                                                 1.2694
   % abs. error                                                    0.4253

Calculations

Linear Regression Equation

      y = 1.0157x – 0.0205

Relative Error of % Protein

      = ((experimental mean – true value) / experimental mean) * 100

      = ((33.925 – 33.5) / 33.925) * 100

      = 1.2694

Absolute Error of % Protein

      = (experimental mean – true value) * 100

      = (33.925 – 33.5) * 100

      = 0.4253
Kjeldahl Equation

       Grams of Protein = grams of Nitrogen * (100g Protein / 15.67g Nitrogen)

                         = .053174 g * (100g Protein / 15.67g Nitrogen) = 0.3393 g Protein

Figure 1 shows the standard curve of the nonfat dry milk samples. The Dye Binding Method

using the protein standard solutions determined this curve. The samples of nonfat dry milk

samples were then analyzed with the spectrophotometer using the dye binding method. The

nonfat dry milk sample %transmittance was used to calculate absorbance (2-log(%trans)).

Concentration is then calculated using the standard curve. These values can be seen in Table

1. The r2 value is 0.99446, which means there is a high correlation between the absorbance

and concentration. Table 2 shows the results from the Dumas Combustion Method. The

percent Nitrogen and Protein can be seen in this table as well as the results for % coefficient

of variation (CV) and % error.



Discussion

       Table 1 shows the results from the analysis and calculations of the Bradford reagent

using the standard curve equation in Figure 1. The % CV was found to be 6.6103 in

absorbance. The % relative error was found to be -1.5493 and the absolute error was found

to be 1.4511. The true value for these calculations was 33.5% because it is the average of the

% of weight/weight of protein of milk powder given in the laboratory handout (Roberts,

2011). The Dumas Combustion Method has a % relative error of 1.2694 and has a %

absolute error of 0.4253 in % protein. This can be seen in Table 2 of the results section. A

smaller absolute error is more accurate. Therefore, the Dumas Combustion Method is more

accurate than the Bradford Reagent assay. Both methods were precise, but the Dumas
Combustion Method had more precision with a %CV of 1.0715 in %Protein. The combustion

method would be assumed to have more accurate and precise results in comparison to using

the reagent because the Dumas instrument would be highly sensitive and would have less

room for error. The Bradford Reagent assay would contain more room for error as students

are transferring the reagents. If the large milk sample was not mixed properly before taking

a smaller sample for analysis, then the results would show a smaller amount of protein.

When the milk was thoroughly mixed, and the experiment was done again, the results

showed an increase two times the amount of protein.

       Accuracy and precision, however, is relative to the standard curve. The accuracy and

precision of methods can only be as accurate and precise as the standard curve. If the curve

has an r2 value close to one, then the methods would probably contain more accurate and/or

precise data. Taking this into account, the Dumas Combustion method would be more

accurate and precise due to the fact that it is not dependent on the standard curve equation.

Though the r2 value was relatively close to 1, the value was not 1. Therefore, the Dumas

Combustion method showed more accurate and precise results.



Questions

1. Non-protein-nitrogen containing substances found in foods include nucleic acids, nitrogen

containing carbohydrates, phospholipids, various alkaloids, vitamins, nitrates and nitrites

(Roberts, 2011). This calculation of the nitrogen content is deemed “crude protein”. The

Dumas Combustion method and the Bradford Reagent method both measure for the crude

protein of the assay. The crude protein is not wanted, but the true protein content of the

sample is wanted for the analysis of nitrogen.
2. The Bradford reagent contains acid in order to denature the proteins for the

determination of the protein. When the proteins are denatured, then the charge is changed.

3. The 12 blank samples are run prior to conducting the analysis in order to determine a

standard curve. If the blanks were not run prior to the analysis of the real samples, then the

atmospheric nitrogen would still be present in the Dumas combustion instrument. This

atmospheric nitrogen contained in the instrument would add to the results of the %nitrogen

content in the sample. Thus, changing the results of the %protein in the sample. Over five

sequential samples, a graph would most likely have similar features as the one shown below.

The graph would have a high percentage of nitrogen to begin with due to the lack of clearing

out the atmospheric nitrogen. The graph would begin to decrease and then plateau.

                       12
                       10
                        8
          % Nitrogen




                        6
                        4
                        2
                        0
                            0   1     2            3        4          5
                                          Trials



Reference

Roberts, Robert. Spring 2011. “Lab 6—Protein Determination.” University Park, PA. 5.
Appendix

Table 3. Protein standard solution analysis using Bradford reagent using a
specrophotometer

                 [BSA]          % Transmittance of the          Absorbance of the Blank
               (mg/mL)                 Blank                        2-log(%trans)
     1              5                    --                                --
     2             2.5                   --                                --
     3            1.25                   --                                --
     4            0.625                  24                             0.6198
     5           0.3125                  44                             0.3565
     6          0.15625                 66.5                            0.1772
     7         0.078125                  81                             0.0915
     8         0.039063                 89.5                            0.0482
     9         0.019531                 97.5                            0.0110

Table 4. Nonfat Milk Dilution analysis using a spectrophotometer
                               % Transmittance of the      Absorbance of the Blank
                                        NFDM                   2-log(%trans)
            1                             49                       0.3098
            2                             46                       0.3372
            3                             44                       0.3565

Table 5. Analysis using Dumas method.
                         NFDM mass             Nitrogen %            Protein %
         1                 0.1993                5.2581               33.547
         2                 0.2054                5.3165               33.919
         3                 0.2054                5.3006               33.818
         4                 0.2040                5.3945               34.417

				
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