LAB REPORT 5

LABORATORY REPORT

SBK3013 PRINCIPLES IN BIOCHEMISTRY

TITLE OF EXPERIMENT : PROTEIN ASSAY

LECTURER : DR ROSMILAH BINTI MISNAN

GROUP : A

GROUP MEMBERS:

HEIDI AMELDA ANAK LAGAT	D20141067086
SHARIFAH AWANIS BINTI SYED MOHD ASWAD	D20141067053
MAUREEN SANTIH ANAK AMBANG	D20141067070

Laboratory 5 : Protein ASSAY

Title: Preparation of Protein Standard

Introduction:

Protein is a macronutrient that necessary for the proper growth and function of our body. Proteins are large biomolecules, or macromolecules, consisting of one or more long chains of amino acid residues. Proteins also perform various functions within organisms, including catalysing metabolic reactions, DNA replication, responding to stimuli, and transporting molecules from one location to another. Our bodies make amino acids in two different ways: Either from scratch, or by modifying others. A few amino acids (known as the essential amino acids) must come from food.

In this experiment, we used two methods to determine the protein concentrations which are absorbance at 540 nm for Biuret and 750 nm for the Lowry assays.

Materials:

1. Protein standard.

2. Biuret reagent

3. Lowry reagent 1 and Lowry reagent 2

4.  8 Test samples:

a)                  Animal samples: chicken, fish, beef

b)                  Plant proteins: soybean, red bean, peanut, green bean, hazelnut

Procedures:

1.                  Preparation of protein Standard

1. Solutions of gelatin at 1, 2, 3, 4, 5, and 6 mg/mL in water from the gelatin stock solution (10 mg/ml) for Biuret assay are prepared.

2. Solutions of gelatin at 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 mg/mL in water from the gelatin stock solution (1 mg/ml) for Lowry method are prepared.

2.                  Preparation of test samples

Procedure:

a.                   Animal protein

1. 10 gram of protein samples (eg. Fish, chicken, beef) are weighed.

2. They are macerated into smaller size.

3. They are blended in Phosphate Buffer saline at 1:10 ratio.

4. Each sample are shake/ stirred for 15 minutes.

5. Each sample is filtered by kitchen filter.

6. Supernatant is collected.

7. The sample is filtered again using Whatman filter No 1

8. Supernatant is collected.

b.                  Plant protein

1. 10 gram of protein samples (eg. Soybean, red bean, peanut, green bean, hazelnut) are weighed.

2. The samples are crushed and grinded into a fine paste or powder using mortar and pestle.

3. The powder is dissolved in Phosphate Buffer saline at 1:10 ratio.

4. The sample is shaken/ stirred for 15 minutes.

5. The sample is filtered by kitchen filter.

6. The supernatant is collected.

7.  The sample is filtered again using Whatman filter No 1.

8. The supernatant is collected.

3.                  Protein Assay:

Procedure:

a.                   Biuret assay

1. All 8 test sample extracts are obtained from the other groups

2. 0.50 mL of each protein (standard and test samples) is mixed with 2.50 mL of Biuret reagent.

3. The absorbance of the samples at 540 nm are measured after 10 minutes.

4. The standard curve are plotted.

5. The protein content of the test sample is estimated using the standard curve.

b.                  Lowry assay

1. All 8 test sample extracts from the other groups are obtained.

2.0.25 mL of each protein (standard and test samples) is mixed with 2.5 mL of Lowry reagent

3. The mixture is incubated at room temperature for 10 minutes.

4. 0.25 mL of Lowry reagent 2 are added and mixed well immediately.

5. The mixture is incubated at room temperature for 30 minutes.

6. The absorbance is measured at 750 nm.

7. The standard curve is plotted.

8. The protein content of the test sample is estimated using the standard curve.

Result:

1.                  Preparation of protein standard

Gelation solution (10 mg/mL of biuret assay ), mg/mL	absorbance
1	0.439
2	0.509
3	0.512
4	0.542
5	0.769
6	0.995

Gelation solution (1 mg/mL of Lowry assay)	Absorbance
0.1	0.132
0.2	0.106
0.3	0.406
0.4	0.256
0.5	0.115
0.6	0.081

a.                   Biuret Assay

Sample	Absorbance	Protein concentration (mg/mL) from graph
Beef	0.488	1.7
Fish	1.020	7.85
Peanut	0.476	1.6
Soybean	0.447	1.1
Chicken	0.552	2.5
Red bean	0.645	3.55
Steamed fish	1.136	9.2
Dhal bean	0.746	4.7
Green bean	0.983	7.4

b. Lowry Assay

TYPES OF SAMPLES	ABSORBANCE (nm)	PROTEIN SAMPLE (mg/mL ) from graph
Beef	0.944	0.470
Fish	1.320	0.540
Peanut	1.219	0.525
Soybean	0.377	0.365
Chicken	1.328	0.545
Red bean	0.926	0.465
Ikan rebus	0.406	0.375
Dal bean	0.214	0.335

a) Biuret Assay

b) Lowry Assay

Discussion

            From this experiment we used Biuret Assay and Lowry Assay to determine the protein content in the samples. In Biuret Assay, we used samples combine with the biuret reagent. Biuret reagent contains copper ions in a basic solution. The copper ions will complex with the amide groups in the proteins to create a blue colour that will be measured using a spectrophotometer. Purpose of Biuret Assay is to prepare the standard curve, to determine the protein content in the sample and to analyse data from standard curve and unknown concentration of the samples.

            In order to determine how much protein concentration is represented by a particular absorbance reading, we need to construct a standard curve. This is done by performing the biuret reaction on a series of prepared solutions of gelatin at 1,2,3,4,5 and 6 mg/ml in water. The absorbance readings obtained from these solutions are used to plot a graph of absorbance as a function of protein concentration called the standard curve for assay. It can be used to determine the protein concentration of the experimental samples (beef, fish, peanut, soybean, red beans, chicken, ikan rebus dan dhal bean.

Based on the graph that has been plotted, it showed that the standard protein concentration for the samples as shown from the result obtained. From the graph we can see that, the highest concentration of protein is ikan rebus (9.2 mg/mL) and the lowest is soybean (1.1 mg/mL). The second highest is fish, 7.85 mg/mL of protein concentration.  The green is the third highest, which is 7.4 mg/mL of protein concentration. Even though the fish is boiled, but the value of protein content remains unchanged. The value of dhal bean, red bean, chicken, beef and peanut are 4.7 mg/mL, 3.55 mg/mL, 2.5 mg/mL and 1.7 mg/mL and 1.6 mg/mL respectively.

The Lowry assay is based on the reaction of cupric ions with peptide bonds under alkaline conditions (the Biuret test). Protein samples are mixed with an alkaline solution containing copper sulphate (Cu2+ ions) which react with peptide bonds to produce Cu+ ions Following this reaction, Folin-Ciocalteau reagent is added where upon the Cu+ ions in the solution react with the Mo(VI) ions to form molybdenum blue – a complex of Mo(IV) and Mo(V) ions. The blue color of the dye can then be measured at an absorance of 750nm. As the amount of Mo(IV) and Mo(V) complex is dependent on the amount of Cu+ ions which is, itself, dependent on the amount of protein in the unknown sample, the color produced is a direct reflection of protein concentration and, with the use of standards, can allow protein concentration to be determined.

Lowry Assay is one of the method that Is used to  determine the total level of protein in a solution. A standard curve is constructed to determine how much protein is represented  by a particular absorbance reading. This is obtained by the Lowry reaction on a series of prepared solutions of gelatin at 0.1,0.2,0.3,0,4.0,5 and 0.6 mg/ml in water. From the series of prepared solutions of gelatin, absorbance(nm) is obtained which is used to construct standard curve (Absorbance vs Protein Concentration). The curve then is used to convert  the absorbance reading for protein samples ( beef,fish, peanut, soybean,chicken,red bean,ikan rebus and dal bean) into protein concentration in the samples.

According to the graph that has been constructed, we able to find the protein concentration in the samples. From the graph, we found that the chicken (0.545 mg/ml) has the highest protein concentration followed by fish (0.540mg/ml) ,peanut (0.525 mg/ml),beef (0.470 mg/ml),red bean (0.465 mg/ml), ikan rebus(0.375), soybean(0.365 mg/ml) and the lowest is dal  bean(0.335 mg/ml).

Questions

1. Describe three alternative methods of determining protein concentration.

a). 280 nm

Aromatic residues, like tyrosine and tryptophan, absorb UV light at 280 nm.  So, if we  have an extinction coefficent for protein, we can measure the absorbance in a UV/Vis spectrometer and calculate the concentration of  protein using Beer’s law (A = elc, where l is the path length of the spectrometer).  We can estimate the extinction coefficient of the protein based on the sequence using Expasy’s ProtParam tool.  Because ProtParam only considers the linear sequence of the protein and doesn’t take into account the structure, which can affect the extinction coefficient, we need to denature the protein before  measure the absorbance.

b) The Bradford assay

It is a colorimetric assay based on the interaction between Coomassie brilliant blue and the arginine and aromatic residues in the protein.  When the dye binds to these residues, its maximum absorption shifts from 470 nm to 595 nm.  In general, we measure the absorbance of a series of known concentrations of a standard protein and create a standard curve. We then use that standard curve to calculate the concentration of your protein sample based on its absorbance.

c) The BCA assay

BCA assay is another colorimetric assay like the Bradford assay.  It makes use of the biuret reaction, in which the protein backbone chelates Cu2+ ions and reduces them to Cu1+ ions.  The Cu1+ ions then react with bicinchoninic acid (BCA) to form a purple-colored product that absorbs at 562 nm.  The procedure is similar to that of the Bradford assay, in which we create a standard curve based on a series of known protein standards.

2. What is an “appropriate blank” and why?

An appropriate blank is control samples, blanks, standards, dummy analyses, and system suitability checks to increase confidence in measurements. Blank is just a solution that does not contain the thing you are measuring, and this gives a base, or zero reading. This helps to ensure the reading is correct.

Conclusion

As a conclusion, there are two ways of measuring protein concentration in a solution. It is either by using biuret assay or Lowry assay.  For biuret assay, ikan rebus contains the highest value of protein concentration. For Lowry Assay, chicken contained the highest value of protein.Both can be used to measure the protein concentration in a solution but the most accurate way to measure the concentration is by using Lowry assay.

Reference

Zhou, P., & Regenstein, J. M. (2006). Determination of total protein content in gelatin solutions with the Lowry or Biuret assay. Journal of food science, 71(8), C474-C479.

Christopher J. Clarke  (2010). Determination of  Protein Concentration by the Lowry Assay. Retrieved from http://www.cst.ur.ac.rw/library/Food%20Science%20books/batch1/Food%20Science%20and%20Technology%20Published%20articles/Analysis%20Protocols/Lowry%20Protein%20Assay.pdf

Hartree-Lowry and Modified Lowry Protein Assays. (n.d.). Retrieved May 21, 2017, from http://www.ruf.rice.edu/~bioslabs/methods/protein/lowry.html

How to Measure Protein Concentration More Accurately. (2011, November 30). Retrieved May 21, 2017, from http://bitesizebio.com/10178/how-to-measure-protein-concentration-more-accurately

Reflection

Heidi

From this experiment, I had learnt how to determine the protein concentration in food samples. Protein is an important component of every cell in the body. The importance of protein for the growth and repair of your muscles, bones, skin, tendons, ligaments, hair, eyes and other tissues is proven since a very long time. This experiment is quite interesting. We conducted the experiment smoothly because each member gave a great teamwork.

Maureen

After the experiment, I get to know the method that can be used to determine the protein concentration in the food. I also get to know that Lowry assay is more accurate than Biuret assay it is quite sensitive and is able to detect even 1 µg of protein. From the experiment, I found that chicken and fish has high concentration of protein compare to other samples. I also learn how to use spectrophotometer to detect the absorbance of food samples. The experiment is quite new and interesting for me.

Awanis

From this experiment, I get to know how to measure the concentration of protein in our daily life food supply. This is an interesting experiment because there are different method and procedure to determine the protein concentration in plant and in animal. From this experiment, we learned both method of it. I actually love the spirit of the teamwork of my team. They never left me to do this experiment and report alone.