Protein Assay Experiment

TITLE: Protein Experiment

Abstracts

From the experiment, we were determined the concentration of protein in different sample of proteins and types of eggs by using absorbance at 540 nm for Biuret and 750 nm for Lowry assays. Biuret reagent will produced violet-purplish color due to the interaction between cupric ions (Cu2+) and peptide bonds under alkaline conditions. While, Lowry reagent produced bluish color due to the reaction of tyrosine and tryptophan residues in proteins. At the end of experiment, Biuret test was showed the positive result for sample proteins and the eggs. The color in Biuret reagent was more obvious compare to Lowry reagent. For the eggs, the result showed that ayam kampung has higher protein concentration compare to other eggs.

Introduction

Proteins are polymers of amino acids. It consist of twenty different types of amino acids occur naturally. Proteins differ from each other according to the type, number and sequence of amino acids that make up the polypeptide backbone. As a result they have different molecular structures, nutritional attributes and physiochemical properties. Proteins are important constituents of foods for a number of different reasons. They are a major source of energy, as well as containing essential amino-acids. Proteins perform a vast array of functions within living organisms, including catalyzing metabolic reactions, replicating DNA, responding to stimuli, and transporting molecules from one location to another. Proteins are also the major structural components of many natural foods, often determining their overall texture such as tenderness of meat or fish products.

To determine protein concentration, Biuret reagent and Lowry reagent are use. Biuret reagent stabilized with sodium potassium tartrate and potassium iodide. A violet-purplish color is produced when cupric ions (Cu2+) interact with peptide bonds under alkaline conditions. The biuret reagent, which contains all the chemicals required to carry out the analysis, can be purchased commercially. It is mixed with a protein solution and then allowed to stand for 15-30 minutes before the absorbance is read at 540 nm. The Lowry method combines the biuret reagent with another reagent (the Folin-Ciocalteau phenol reagent) which reacts with tyrosine and tryptophan residues in proteins. This gives a bluish color which can be read somewhere between 500 - 750 nm depending on the sensitivity required. This method is more sensitive to low concentrations of proteins than the biuret method.

Procedure

Biuret Test

1.      The standard protein solutions are prepared at 1, 2, 3, 4, 5, and 6 mg/mL in water.

2.      The sample proteins of albumin are prepared at 0.1% of Ayam Kampung’s egg, 0.1% of Ayam Omega 3’s egg, 0.1% of normal chicken egg, 0.1% of Puyuh’s egg, and 0.1% of duck’s egg.

3.      All 0.5 mL of these standard proteins and sample proteins of albumin are mixed with completely with 2.5 mL of Biuret reagent.

4.      Finally, the absorbances of these samples are measured at 540 nm after 10 minutes.

Lowry Test

1.      The standard protein solutions are prepared at 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 mg/mL in water.

2.      The sample proteins of albumin are prepared at 0.1% of Ayam Kampung’s egg, 0.1% of Ayam Omega 3’s egg, 0.1% of normal chicken egg, 0.1% of Puyuh’s egg, and 0.1% of duck’s egg.

3.      All 0.25 mL of these standard proteins and sample proteins of albumin are mixed with completely with 2.5 mL of Lowry reagent 1.

4.      After 10 minutes, they are added with 0.25 mL of Lowry reagent 2 and mix them well immediately.

5.      Finally, the absorbances of these samples are measured at 750 nm after 30 minutes.

 Results (Biuret Assay)

Sample Protein	Absorbance (nm)
Puyuh	0.193
Itik	0.219
Ayam Omega 3	0.206
Ayam Kampung	0.246
Ayam BIasa	0.218

Dilution factors = 10x 5

                                = 50ml

Total protein = density of sample solution (mg/ml) x dilution factor (ml)

Type of protein	Total protein  (mg)
Ayam kampung	106
Ayam biasa	96.5
Itik	103.5
Ayam omega	83.5
Puyuh	56

Discussions:

            This experiment was carried out to determining protein concentration. Usually in biology we always carry out Biuret test to detect the presence of protein using Biuret’s reagent. In biochemistry we tend to run quantitative measurement experiment. As this protein assay experiment, we used Biuret assay and Lowry assay. Based on the hypothesis, the high its absorbance reading, the more concentrated its protein concentration.  As we know, Lowry assay is more effective than Biuret assay because even with low concentration of protein, the reading of its absorbance can be obtained by using Lowry assay. However, for the results, we only shows the result for Biuret assay on protein concentration of 5 different of bird’s albumin. This is because there are errors while doing Lowry assay which the outcome for this test are not valid. The error can be because of the mixing uses of pipette during the prepared the solutions.

            Based on the result given for Biuret Test, “ayam Kampung” has the highest protein concentration with total protein of 106 mg while the lowest is Puyuh.with total protein of 56 mg. This assay helps us to determine the standard protein estimation in all the types of egg we tested. However, when it comes to determine total protein concentration of a sample, one must first select an appropriate protein assay method based upon its compatibility with the samples type. The objective is to select a method that requires the least manipulation or pre-treatment of the samples to accommodate substances that interfere with the assay. Each method has its particular advantages and disadvantages. Because no one reagent can be considered the ideal or best protein assay method for all circumstances, most researchers have more than one type of protein assay available in their laboratories.

Important criteria for choosing an assay include:

• Compatibility with the sample type and components
• Assay range and required sample volume
• Protein-to-protein uniformity (see below)
• Speed and convenience for the number of samples to be tested
• Availability of spectrophotometer or plate reader necessary to measure the color produced (absorbance) by the assay

With most protein assays, sample protein concentrations are determined by comparing their assay responses to that of a dilution-series of standards whose concentrations are known. Protein samples and standards are processed in the same manner by mixing them with assay reagent and using a spectrophotometer to measure the absorbances. The responses of the standards are used to plot or calculate a standard curve. Absorbance values of unknown samples are then interpolated onto the plot or formula for the standard curve to determine their concentrations. Obviously, the most accurate results are possible only when unknown and standard samples are treated identically. This includes assaying them at the same time and in the same buffer conditions, if possible. Because different pipetting steps are involved, replicates are necessary if one wishes to calculate statistics (e.g., standard deviation, coefficient of variation) to account for random error. Although most modern spectrophotometers and plate readers have built-in software programs for protein assay data analysis, several factors are frequently misunderstood by technicians. Taking a few minutes to study and correctly apply the principles involved in these calculations can greatly enhance one's ability to design assays that yield the most accurate results possible. (Retrieved from http://www.piercenet.com/browse.cfm?fldID=BE219700-9B95-43A1-A3DA-83800F1A0392#select-assay )