estimination of protein 2

TABLE OF CONTENT

No. Title pages

1 Abstract/Summary 2

2 Introduction 3

3 Aims 4

4 Theory 4

5 Methodology/Procedure 5

6 Appatatus 7

7 Results 9

8 Calculations 10

9 Discussion 16

10 Conclusion 17

11 Recommendations 17

12 Reference 18

13 Appendices 18

1

ABSTRACT

This experiment is conducted to learn the principles of protein assays. This exercise

introduces students to method of determining protein concentrations by using several method

which are lowry method, Bradford method and Biuret method.. This lab activity is designed

to teach students the principles behind a common protein estimation assay and identify the

efficiency and sensitivity of these three method of estimination of proteins. In this

experiment, there are two types of proteins have been used which are gelatine and bovine

serum albumin (BSA)

Standard Biuret reagent is prepared. The 0.50mL of protein is mixed with the

prepared Biuret Reagent. The solution is mixed well and allows standing for 10 minute. The

absorbance for each tube is read against the blank at 540 nm. The standard curve is plotted

using concentration of standard (mg/mL) against the absorbance at 540 nm.

Protein assays based on Lowry methods are divided into two categories, dye binding

protein assays and protein assays based on alkaline copper. Under alkaline conditions cupric

ions (Cu2+) chelate with the peptide bonds resulting in reduction of cupric ions (Cu2+) to

cuprous ions (Cu). The Cuprous ions can also be detected with Folin Ciocalteu Reagent

(phosphomolybdic/phosphotungstic acid) and this method is commonly referred to as the

Lowry method. Cuprous ions (Cu+) reduction of Folin Ciocalteu Reagent produces a blue

color that can be read at 750nm. The amount of color produced is proportional to the amount

of peptide bonds. In this experiment ,0.5 ml of protein is added to 5 ml of lowry reagent #1

and after 10 min,lowry reagent #2 is added and mix well. The mixture is leave for 30

minutes before using it

For Bradford method it is relies on the binding of Coomassie Brilliant Blue G250 dye

to protein, in which protein concentration is proportionate to the dye. In this experiment, 0.5

ml of protein with different concentration which are 0.02,0.04,0.06,0.08,0.10,0.12,0.14 and

0.16 are diluted with 5 ml of Bradford reagent The solution is mixed well and allows standing

for 10 minute. The absorbance for each tube is read against the blank at 595 nm. The standard

curve is plotted using concentration of standard (mg/mL) against the absorbance at 595 nm.

2

Sensitivity is measured on how well does it detect small amounts of protein; for

instance, what mass of protein is needed to give an absorbance of 0.01The sensitivity increase

as the amount of protein detected increase. Based on this experiment, the most efficient and

sensitive is Bradford method because it has the highest amount of protein. The amount of

protein for gelatine is 41.82 mg while for BSA is 38.6 mg. The second efficient and sensitive

is Biuret method which the amount of protein for gelatine is 5.72 mg while for BSA is 14.1

mg. The lest sensitive and efficient method is Lowry method because contain the lowest

amount of protein. The amount of protein for gelatine is mg while for BSA is 38.6 mg. The

result for Biuret and Lowry is slightly different from the theory, this happen might because

of some error

INTRODUCTION

The protein in solution can be measured quantitatively by different methods. The methods

have been used in this experiment are Biuret method, Bradford method and Lowry method.

And in this experiment there are two types of protein have been used which are bovine serum

albumin (BSA) and gelatine.

The methods described by Bradford uses a different concept-the protein‘s capacity to bind to

a dye, quantitatively. The assay is based on the ability of proteins to bind to coomassie

brilliant blue and form a complex whose extinction coefficient is much greater than that of

free dye.

Protein assays based on Lowry methods are divided into two categories, dye binding protein

assays and protein assays based on alkaline copper. Under alkaline conditions cupric ions

(Cu2+) chelate with the peptide bonds resulting in reduction of cupric ions (Cu2+) to cuprous

ions (Cu). The Cuprous ions can also be detected with Folin Ciocalteu Reagent

(phosphomolybdic/phosphotungstic acid) and this method is commonly referred to as the

Lowry method.

For biuret method,In alkaline solutions, cupric ion complexes with the peptide bonds of

proteins and peptides to form a purple charge transfer complex (λmax = 540 nm). The

intensity of the color is proportional to the protein concentration. This reaction occurs only

with the peptide bond and not with the amino acid side chains. Because the number of

peptide bonds per given unit weight is approximately the same for all proteins, this method is

3

generally applicable and reasonably accurate, irrespective of the composition of the protein

mixture

AIMS

This experiment is conducted:

1. To learn the principles of protein assays.

2. To determine protein concentrations by using several method which are lowry

method, Bradford method and biuret method..

3. To identify the efficiency and sensitivity of these three method of estimination of

proteins

THEORY

Biuret method determination of protein determination

In alkaline condition ,copper (II) is thought to bind to the peptide nitrogens of proteins and

this complex absorb light maximally at 550nm.in addition ,it is thought that in biuret reaction

some of the Cu²+ is reduce to Cu + in a secondary reaction with proteins

Figure 1 putative cupric complex with peptide bond of proteins

Lowry method of protein determination

The lowry method is commonly used method of protein determination because it is

inexpensive, easy to perform , sensitive, and highly reproducible Lowry is based on copper

complex formation with the peptide bond. Lowry methods are divided into two categories,

dye binding protein assays and protein assays based on alkaline copper. Under alkaline

4

conditions cupric ions (Cu2+) chelate with the peptide bonds resulting in reduction of cupric

ions (Cu2+) to cuprous ions (Cu). The Cuprous ions can also be detected with Folin Ciocalteu

Reagent (phosphomolybdic/phosphotungstic acid) and this method is commonly referred to

as the Lowry method. Cuprous ions (Cu+) reduction of Folin Ciocalteu Reagent produces a

blue color that can be read at 750nm. The amount of color produced is proportional to the

amount of peptide bonds

Bradford method of protein estimination

The methods described by Bradford uses a different concept-the protein‘s capacity to bind to

a dye, quantitatively the one of the most commonly used method of protein determination

because it is easy to perform , very sensitive. The assay is based on the ability of proteins to

bind to coomassie brilliant blue and form a complex whose extinction coefficient is much

greater than that of free dye. Bradford is influenced by pH of your protein solution (not only

due to the protonation state of your charge residue side chain but also the dye has different

absorbance maxima at different pH).

METHODOLOGY

Preparation of stock solution

1. The gelatin or bsa which in form of powder is dilute with distilled water to form

stock solution of 0.16 mgml .

2. the mass of powder need to be added is calculated by ,

stock solution =mass of powder

Volume of water

Preparation of Lowry reagent

Lowry reagent #1

One volume of 500 ml (2.0 % sodium carbonate and 0.4% sodium hydroxide) is

diluted with 50 volume of 10 ml (0.5 % copper sulphate and 1.0 %sodium tartarate)

5

Lowry reagent # 2

5 ml of folin abcatteu phenol is diluted with 5ml of distilled water

Preparation of blank sample

1. Lowry assay: o.5 ml of distilled water is diluted with 5 ml of lowry reagent 1 .then,

after 10 minutes, 0.5 ml of lowry reagent 2 is added and mixed well.leave it for 30

minutes before using it.

2. Biuret assay : one ml of distilled water is diluted with five ml of biuret reagent

3. Bradford assay : 0.5 ml of distilled water is diluted with five ml of Bradford reagent.

Biuret method

1. The stock solution either BSA sample or gelatin is diluted with distilled water for the

concentration 0.02,0.04,0.06,0.08,0.10,0.12,0.14,0.16.

2. The amount of distilled water need to be added to the stock solution was calculated

by using this formula

m1 v1=m2 v2

3. 1.0 ml of sample is diluted with 5.0 ml of biuret reagent

4. The solution is mixed well and allows standing for 10 minute

5. The absorbance for each tube is read against the blank at 540 nm

6. The standard curve is plotted using concentration of standard (mg/mL) against the

absorbance at 540 nm

Bradford method


concentration 0.02,0.04,0.06,0.08,0.10,0.12,0.14,0.16.



m1 v1=m2v2

3. 0.5 ml of sample is diluted with 5.0 ml of Bradford reagent


6




Lowry method


concentration 0.02,0.04,0.06,0.08,0.10,0.12,0.14,0.16.



m1 v1=m2v2

3. 0.5 ml of sample is diluted with 5.0 ml of Lowry reagent #1


5. After 10 minutes0.5 ml of Lowry reagent #2 is added

6. The solution is leave for 30 minutes




APPARATUS

Proteins: bovine serum albumin (BSA) and gelatine

Distilled water

Biuret Reagent

1. 10% (w/v) NaOH

2. 0.3% copper sulfate pentahydrate

3. 1.2% sodium potassium tartrate

4. potassium iodide

Lowry Reagents

1. Reagent 1:

7

Reagent B (0.5% copper sulfate pentahydrate, 1% sodium or potassium tartrate)

Reagent A (2% sodium carbonate, 0.4% NaOH)

2. Reagent 2:

Folin-Ciocalteu phenol reagent

Bradford's Reagent

1. Coomassie Blue G-250

2. 95% ethanol

3. 85% phosphoric acid

8

RESULT

gelatin sample

Concentration of

gelatine mg/ml

Method

Biuret assay Bradfort assay Lowry assay

0.02 0.032 0.480 0.852

0.04 0.042 0.607 0.705

0.06 0.034 0.507 0.886

0.08 0.039 0.705 1.114

0.10 0.033 0.713 2.037

0.12 0.046 0.740 0.920

0.14 0.032 0.623 0.681

0.16 0.036 0.621 3.300

BSA sample

Concentration of

gelatine

Method

Biuret assay Bradfort assay Lowry assay

0.02 0.034 1.518 1.090

0.04 0.043 1.610 1.674

0.06 0.044 1.655 2.104

0.08 0.046 1.689 2.508

0.10 0.050 1.704 2.695

0.12 0.051 1.723 3.002

0.16 0.054 1.762 3.252

0.16 0.052 1.763 3.073

9

CALCULATION

Biuret method in gelatine sample

0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.180

0.0050.01

0.0150.02

0.0250.03

0.0350.04

0.0450.05

f(x) = 0.161904761904762 x + 0.0209166666666667

Absorbance at 540 nm vs Concentration of mixture

Y-ValuesLinear (Y-Values)

concentration of sample (mg/ml)

Absobance at 540 nm

Y= mx + c, where y=0.01

Y=0.1619x +0.0209

X=0.01-0.0209

0.1619

=0.0673 mg/ml

Amount of protein

=0.0673 mg/ml x 85ml

=5.72mg

10

Bradford method in gelatine sample

0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.180

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

f(x) = 1.05595238095238 x + 0.529464285714286




Absobance at 595 nm


Y=1.056x +0.5295

X=0.01-0.5295

1.056

=0.492 mg/ml

Amount of protein

=0.492 mg/ml x 85ml

=41.82 mg

11

Lowry method in gelatine sample

0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.180

0.5

1

1.5

2

2.5

3

3.5

f(x) = 9.13095238095238 x + 0.395464285714286




Absobance at 750 nm


Y=9.131x +0.3955

X=0.01-0.3955

9.131

=0.0422 mg/ml

Amount of protein

=0.0422 mg/ml x 85ml

=3.95 mg

12

Biuret method in bsa sample

0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.180

0.01

0.02

0.03

0.04

0.05

0.06f(x) = 0.146428571428571 x + 0.0342857142857143




Absobance at 540 nm


Y=0.1464 x +0.0343

X=0.01-0.0343

0.1464

=0.1659 mg/ml

Amount of protein

=0.1659 mg/ml x 85ml

=14.1 mg

13

Bradford method in bsa sample

0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.180

0.20.40.60.8

11.21.41.61.8

2

f(x) = 3.10357142857143 x + 1.35367857142857




Absobance at 595 nm


Y=3.1036x + 1.3537

X=0.01-1.3537

3.1036

=0.433 mg/ml

Amount of protein

=0.433 mg/ml x 85ml

=36.8 mg

14

Lowry method in bsa sample

0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.180

0.5

1

1.5

2

2.5

3

3.5f(x) = 17.3803571428571 x + 0.941714285714286




Absobance at 750 nm


Y=17.38x +0.9417

X=0.01-0.9417

17.38

=0.0536 mg/ml

Amount of protein

=0.0536 mg/ml x 85ml

=4.557 mg

15

DISCUSSION

This experiment is done to apply the general approach which is to determine the

amount of protein by using several method which biuret method ,Bradford method and lowry

method, and to identify the efficiency and sensitivity by using three method of estimination

of protein. There are two types of proteins have been used which are gelatine and bovine

serum albumin (BSA)

In this experiment, the graph of absorbance at 540nm versus concentration of

sample(mg/ml) for Biuret, the graph of absorbance at 595 nm versus concentration of

sample (mg/ml) for Bradford and graph of absorbance at 750 nm versus concentration of

sample (mg/ml) for Lowry are plotted for both protein. Based on this experiment for the

generality ,the result among two different proteins are consistent because the result for BSA

sample and gelatine quite similar for Bradford method and Lowry method, but for Biuret the

result among the protein is not consistence because the result quite different which for

gelatine sample is 5.72 mg while for BSA sample is 14.1 mg. For the linearity ,all the graph

gives a straight line, but for the gelatine sample even though the graph gives the straight line

but some of the point is alternating for Biuret method while for Lowry method and Bradford

method the point quite not consistent.

For sensitivity based on the theoretical states that the most sensitive method is

Bradford method followed by the Lowry method and the less sensitive is Biuret method.

Sensitivity is measured on how well does it detect small amounts of protein; for instance,

what mass of protein is needed to give an absorbance of 0.01The sensitivity increase as the

amount of protein detected increase. Based on this experiment, the most efficient and

sensitive is Bradford method because it has the highest amount of protein. The amount of

protein for gelatine is 41.82 mg while for BSA is 38.6 mg. The second efficient and sensitive

is Biuret method which the amount of protein for gelatine is 5.72 mg while for BSA is 14.1

mg. The lest sensitive and efficient method is Lowry method because contain the lowest

amount of protein. The amount of protein for gelatine is mg while for BSA is 38.6 mg. The

result for Biuret and Lowry is slightly different from the theory, this happen might because

of some error .

16

There are several source of error need to be consider .first, parallax error occur while

carrying this experiment because the eye is not perpendicular to the readings of measuring

cylinder. Second, the reagents expose to the air because some of the reagents are volatile.

Next, using the unclean and contaminate apparatus that make the result less accurate

especially for cuvettes because the spectrophotometer is really sensitive. Besides, using the

unspecific tissue while wiping the cuvette, this can make some particle stick to the cuvette

since the spectrophotometer is sensitive.

CONCLUSION

As conclusion, this experiment is carryout successfully. The objective which to determine the

amount of protein by using several method which Biuret method ,Bradford method and

Lowry method ,and to identify the efficiency and sensitivity by using three method of

estimination of protein are achieved. Based on this experiment, Bradford method is the most

sensitive and efficient method since it can detect large amount of protein at absorbance 0.01

for BSA and gelatine sample. The amount of protein detected for gelatine is 41.82 mg while

for BSA is 36.8 mg. Unfortunately for Biuret method and Lowry method is slightly different

from the theoretical this might be due to some error while carrying this experiment.

RECOMMENDATION

Use the clean apparatus to ensure the result of the experiment is not disturb by

unknown solution and to prevent from the contamination

Avoid parallax error and make sure the eyes is perpendicular to the readings of the

measuring cylinder

Ensure that to handle carefully cuvette bottle for accuracy and prevent contamination.

We have to handle cuvette with only gloves and touch only the areas not in the light

path.

Ensure that using specific tissue while wiping the cuvettes to get the accurate result

Besides that, the mixture of sample of either gelatin or bsa are dilute with water

properly and both are shaken for several times to make sure the homogenous mixture

formed.

ensure that there is no air bubbles trapped in pipette because it can make the result

less accurate.

REFERENCES

17

1. Gornall, A.G., Bardawill, C. J. and David, M. M. (1949)Determination of

Serum Proteins by means of the Biuret Reaction.J. Biol. Chem. 177; 751.

2. Layne, Ennis Spectrophotometric and Turbidimetric Methods for Measuring

Proteins.Methods in Enzymology, vol. 3, p. 447 (1957)

3. Peterson, Gary L.Determination of Total Protein.Methods in Enzymology,

vol. 91, p. 95 (1983)

APPENDICES

Figure 2:gelatin sample with different concentration

Figure 3:Bradford method

18

estimination of protein 2

Documents