Monday 8 April 2013

FUN EXP 2: Protein Assay



ABSTRACT

For this experiment, we shows to you about the protein content in the egg by using two different tests which are Lowry assay and Biuret reagent. The type of egg that we study are Telur Ayam Biasa, Telur Ayam Kampung, Telur Itik, Telur Puyuh and Telur Omega 3.Before that, we need to identify the standard concentration foe each indicator which are Lowry and Biuret reagent. After plotting into the graph, we can conclude that for Lowry reagent, the highest concentration of protein is Ayam Biasa which is 0.6 mg/L, followed by Telur Ayam Kampung 0.56mg/L , Telur Itik 0.54 mg/L , Puyuh 0.5 mg/L and Omega3  0.41 mg/L. For Biuret reagent, Telur Omega3 have the highest protein content which is 6.0 mg/L followed by Telur Ayam Kampung 2.60mg/L , Telur puyuh 2.35 mg/L, Telur Itik 2.25mg/L and telur Ayam biasa is 1.4 mg/L. 


Introduction:

            The determination of protein concentration is an essential technique in all aspects of protein studies. Each protein assay method has its own advantages and limitations and often it is necessary to obtain more than one type of protein assay for study. Protein assays based on these methods are divided into two categories: dye binding protein assays and protein assays based on alkaline copper.

            In this experiment we investigate the protein assay based on alkaline copper which is Biuret Assay and Lowry Assay. For Biuret assay, the reaction of protein with the copper ions at a basic pH to become complex is observed. When this reaction is used to measure protein concentrations, it is called the Biuret Protein Assay. The copper ions will complex with the amide groups in the proteins to create a blue color that will be measured using a spectrophotometer. The amount of blue color that forms is directly proportional to the quantity of protein in our samples. The interaction of cupric ions (Cu2+) with protein results in a blue color that can be read at 545nm.

        For the Lowry assay, under alkaline conditions cooper ions react with peptide bond resulting in reduction of cupric ions (Cu2+) to cuprous ion (Cu+). The Cuprous ions can be detected by of the Folin-Ciocalteay reagent (phosphomolybdic/phosphotungstic acid). The amount of color produced is proportional to the amount of peptide bonds. Cuprous ions (Cu+) reduction of Folin Ciocalteu Reagent produces a dark color that can be read at 650-750nm.  This experiment will demonstrate the Biuret assay for proteins concentration determination at absorbance 540 nm while Lowry assay at absorbance 750nm. 


BIURET REAGENT

Apparatus and materials:
  • ·         Test tubes
  • ·         Cuvettes and spectrophotometer
  • ·         Pipette
  • ·         Biuret reagent
  • ·         Proteins sample


Procedures :


The solutions of gelatin was prepared at 1,2,3,4,5 and 6 mg/ml in water for Biuret assay.


0.50 ml of protein was mixed with 2.50ml of Biuret reagent.


The absorbance of the standard Biuret reagent was measured after 10 minutes by using spectrophotometer.


The reading of the absorbance was recorded and a standard curve was plotted.


0.1ml of pure protein samples which are consists of telur itik,telur puyuh,telur ayam biasa,telur ayam kampung and telur omega was diluted with 19.9ml of distilled water.


0.50 ml from each of protein samples were mixed with 2.50ml of Biuret reagent and left for about 10 minutes.

                                                                             
After 10 minutes, the reading of all samples were measured by using spectrophotometer and recorded in the table provided.



The standard curve for the protein samples was plotted and the standard solution for each of the samples was determined.


The concentration of each proteins sample were compared by using both method.





Results :

     Standard solution of Biuret assay (mg/mL)
      Absorbance of 1mL at 540 nm
     1   
      0.134 nm
     2
      0.164 nm
     3
      0.199 nm
       4                                                                   
      0.278 nm
    5
      0.269 nm
    6
      0.356 nm

  Table 1: The absorbance of 1mL of standard solution of Biuret assay at 540 nm.

      Protein sample
     Absorbance of 1mL at 540 nm
      Telur itik
      0.180 nm
      Telur puyuh
      0.182 nm
      Telur ayam biasa
      0.137 nm
      Telur ayam kampung
      0.195 nm
      Telur omega
     0.356 nm

Table 2: The absorbance of 1mL of protein sample at 540 nm



Dicussion :

From the graph that we had plotted, we can found that the standard solution of Biuret assay of the proteins sample :
a)      Standard solutions of Biuret assay for telur itik is 2.25 mg/ml x 400
b)      Standard solutions of Biuret assay for telur puyuh is 2.35 mg/ml x 200
c)      Standard solutions of Biuret assay for telur ayam biasa is 1.4 mg/ml x 200
d)     Standard solutions of Biuret assay for telur ayam kampung is 2.60 mg/ml x 200
e)      Standard solutions of Biuret assay for telur omega is 6.0 mg/ml x 10


LOWRY REAGENT

Procedure:

  1.     0.25 mL of protein is mixed with 2.5 mL of Lowry reagent.
  2.     After 10 minutes, 0.25 mL of Lowry reagent 2 added and mixed well immediately.
  3.     After 30 minutes, the absorbance at 750 nm is measured and standard curve is plotted.
Lowry reagent 1:
1.       One volume of reagent B is mixed (0.5% copper sulfate pentahydrate, 1% sodium of potassium tartrate) with 50 volumes of reagent A (2 % sodium carbonate, 0.4% NaOH). Bothe reagent A and B are supposed to be stable for a long time)
Lowry reagent 2:
1.       Commercial Folin- Ciocalteu reagent is diluted with an equal volume of water. Its stable for a few days or weeks.
All series should include a zero protein (water) tube (reagent blank) 


Result:

S      Standard
         Concentration (mg/mL)
                                          Absorbance (750 nm)
      Blank
      Standard
      0.1
      0.130
     0.271
     0.2
      0.125
     0.160
     0.3
     0.125
     0.178
     0.4
     0.127
     0.217
     0.5
     0.125
     0.204
     0.6
     0.124
     0.296

         Unknown Concentration
     Unkonwn Concentration (mg/mL)
                              Absorbance (750 nm)
       Blank
        Protein
      Telur ayam kampung
      0.118
     0.280
      Telur ayam biasa
     0.122
     0.296
      Telur itik
     0.122
     0.271
     Telur puyuh
     0.124
     0.262
     Telur omega 3
     0.127
     0.222


         

      
         Discussions:

        Lowry assay

        Biochemical assay for determining the total level of protein in a solution. Oliver H. Lowry is the person who create the reagent responsible for protein level determination.
  This method was prepared under alkaline conditions. The divalent copper ions will forms a complex with peptide bonds in which it is reduced to a monovalent ion.


This means that the aromatic amino acids in the treated sample reduce the phosphomolybdatephosphotungstic acid present in the Folin Reagent (Folin-Ciocalteu phenol reagent in Lowry reagent 2). Thus, this may result in the blue color of the solution.

  1. From the experiment, with referring to the standard that we have done, we can see that “telur ayam kampung” shows 0.280nm which is near to the absorbance of 0.6 mg/mL concentration of the standard.
  2. Meanwhile “telur ayam biasa” has 0.296nm absorbance, which shows the same absorbance reading of the 0.6mg/mL concentration standard.
  3. Most of the eggs showed absorbance reading near to 0.296nm which means that they may contain 0.6 mg/mL protein. Meanwhile the duck egg shows that it only have 0.1 mg/mL protein by comparing its absorbance and the standard.
  4. The Lowry method very sensitive to a lower concentration of protein down to about 0.01 mg/mL. This method is best used on solutions with concentration in the range 0.01-1.0mg/mL of protein.
  5. Lowry method has a disadvantage. It only accurate when the protein is free from non-protein agents such as buffers, drugs, nucleic acids and sugars. That is why we need to dilute them so that the agents can be minimized in assuming that the protein concentration is sufficiently high and can be detected after the dilution.
    Thus, when the interfering agent are involve, we need to run appropriate blank. Appropriate blank is the cuvette without a standard protein which is made up in the same agent as in the sample. This way, if the agent has an effect on the protein assay reagent, each sample and the blank will have been altered the same. The blank is used to set the instrument to the zero absorbance.
  1. Other three alternative methods of determining protein concentration is by using Enzyme-linked immunobsorbent assay (ELISA), the Bicinchonicic Acid (BCA) assay and Protein 200 plus assay
  2. ELISA - involves the immobilization of the antigen/protein of interest. There are two  types of ELISA assay; direct and indirect. The direct ELISA assay is faster compared to indirect ELISA assay and eliminates the problems of cross-reactivity of secondary antibody.
  3. BCA assay - similar to Biuret reagent and Lowry protein assay. It can determine total concentration of protein range in 0.5 µg/mL to 1.5 mg/mL.. This method can be applied in higher temperature  (37-60 oC) to increase the assay sensitivity while minimizing the variances caused by unequal amino acid composition.
  4. Protein 200 plus assay- The chip-based were performed on the Agilent 2100 bioanalyzer in combination of Protein 200 Plus LabChip kit. All chips prepared according to the protocol provided with the 200 Plus LabChip kit. The Agilent 2100 bioanalyzer in combination with the Protein 200 Plus LabChip kit and the absolute quantification feature of the sowftware provides fast and reliable accurate data.
     Conclusion:
      In conclusion, the biuret and lowry reagent can be used to determine the protein content in the egg. The higher concentration of protein is been found in Omega 3 egg.

     References:






















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