lab exercise 2 - fish muscle protein (autosaved).docx

7/28/2019 Lab Exercise 2 - Fish Muscle Protein (Autosaved).docx

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Exercise 2Fish Muscle Protein

IntroductionThe protein content of fish muscle varies with species, season and the environments.High levels of protein are found in the feeding around the spawning season. There are

obvious incentives in using fish with high protein content.

The protein in the sarcoplasmic fraction is excellently suited to distinguish fish species,as each species has a characteristic band run when separated by isoelectric focusingmethod or electrophoresis. For example, one might be able to use this technique todetect the substitution of inexpensive fish for an expensive fish.

In this experiment, sarcoplasmic muscle proteins are extracted with a 0.15 M saltsolution, the protein content of the extract is measured by the BCA assay. In which theprotein present reduces cupric ions to cuprous ions under alkaline conditions. Thecuprous ions react with the BCA reagent to give a purple colour that can be quantified

spectrophotometrically and related to the protein content. (Suzanne, 2010)

ObjectiveTo extract the protein from the muscles of patin fish (freshwater) and siakap fish(saltwater) and measure the protein content of the extracts.

MaterialsSAMPLE EXTRACTIONSodium chloride, NaClSodium phosphate, monobasic (NaH2PO4.H2O)Blender

1 x 200 ml beaker1 x 15 ml centrifuges tubes1 x funnel1 x test tubeFilter paperPatin and Siakap Fish

PROTEIN DETERMINATION (BCA Assay Kit)Kit Contents :BCA Reagent A, 500 mlBCA Reagent B, 25 ml

Albumin Standard Ampules, 2 mg/ml, 10 x 1 mlCentrifuges tubesMicropipette tips37

oC waterbath

CuvettesSpectrophotometer


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MethodPart 1 : Preparation of Reagent and Sample Processing

Sample Extraction Buffer1. 500 ml of extraction buffer was prepared which contain 0.15 M sodium chloride

and 0.05 M sodium phosphate at pH 7.0

Sample Preparation1. 40g of fish muscle were blended in 120ml of extraction buffer.2. 10 ml of the muscle homogenate was poured into 15 ml centrifuge tube.3. The sample is centrifuged at 2000 x g for 15 minutes at room temperature.4. The supernatant is filtered using Whatman No.1 filter paper and collected in a

test tube and capped.5. The filtrate is stored at 4

oC for 1 week.

Part 2 : Analysis

BCA Protein Assay1. The Working Reagent for the BCA assay was prepared by combining Pierce

Reagent A with Pierce Reagent B, 50:1 (v/v), A:B. 10 ml of Reagent A and 0.2 mlReagent B were used to prepare 10.2 ml of Working Reagent.

2. Dilution of 1:10 and 1:20 of supernatant was prepared in extraction buffer in afinal volume of 1ml

3. Duplicates were prepared for each reaction mixture of diluted extracts and BSAstandards as indicated in the table below.

4. Each reaction mixture was mixed with a vortex mixer, and then incubated in awater bath at 37C for 30 min.

5. The absorbance of each tube at 560nm were obtained and recorded.

Dilution Fish extract Extraction Buffer

1: 10 ml 100 l 0.9 ml 900 l

1: 20 50 l 950 l

Tube Identity ddH2O BSA std(l)

Fish extract(l)

Working reagent(ml)

Blank 50 0 - 0

Std 1 45 5 - 0.142

Std 2 40 10 - 0.261

Std 3 20 30 - 0.990

Std 4 10 40 - 1.659

Std 5 0 50 - 0.907

Sample 1:10 25 - 25 0.485

Sample 1:20 25 - 25 0.129


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6. A standard curve was plotted, absorbance at y-axis and protein concentration onx-axis. Equation of the line for the standard curve was determined.

7. The protein concentrations were calculated based on the equation of the line.

Results

PATIN FISH

Tube identityddH2O

(l)BSA Std

(l)

BSAconcentration

(mg/ml)

Absorbance(O.D)

Blank 50 0 0 0S1 45 5 0.2 0.142

S2 40 10 0.4 0.261

S3 20 30 1.2 0.990S4 10 40 1.6 1.659S5 0 50 2.0 0.907

Sample 1:10 25 - 25 0.485

Sample 1:20 25 - 25 0.129

SIAKAP FISHConcentration of fish protein:1:10 = 5.546 mg/mL1:20 = 4.684 mg/mL


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Graph

Graph of BSA Concentration against Absorbance (560nm)

Equation of line y = 0.9704x + 0.0876

CalculationConcentration of

a) Sample 1:10Absorbance value, x = 0.485 x 10Concentration = 0.9704(4.85) + 0.0876

= 5.582 mg/ml

b) Sample 1:20Absorbance value, x = 0.129 x 20

= 2.58Concentration = 0.9704(2.58) + 0.0876

= 4.256 mg/ml

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8

BSAConcentration(mg/ml)

Absorbance (O.D)


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Discussiona) In BCA analysis, Proteins reduce alkaline Cu (II) to Cu (I) in a concentration

dependent manner. Bicinchoninic Acid (BCA) is a highly specific chromagenicreagent for Cu (I) forming a complex with an absorbance maximum at 560 nm.Because of this property, the resultant absorbance at 560 nm is directlyproportional to the protein concentration (Aldrich, 2013)

b) In BCA analysis, two main steps are involved. First is the biuret reaction, whereblue colour formed which is due to the reduction of Cu2+ to Cu+. Second is thechelation of BCA with the cuprous ion (Cu+),where purple color is formed. Thepurple coloured reaction product is formed by the chelation of two molecules ofBCA with one cuprous ion (Thermo Scientific, 2013).

c) The concentration of fish muscle against the absorbance value at 560nm showsa proportional increase. This graph is plotted with the values of absorbance 5standard solution and blank as a control. By plotting the graph, linear regressionline can be obtained which is used to calculate the sample fish concentration.

From the calculation, we have 5.582 mg/ml for 1: 10 dilution and 4.256 mg/ml.During the calculation, the x value is multiplied by 10 and 20 which are thedilution factors.

d) Two dilution of fish extract was prepared so that we could get an absorbancereading that is within a range of the protein concentration

e) From our analysis, the protein concentration of patin fish is higher in 1:10 dilutionand lower in 1:20 dilution compared to siakap fish. Each fish have differentprotein concentration and in this case, the protein concentration of 2 fish samplediffer may due to season and the environmental condition. There might be

different in the protein concentration of saltwater fish and freshwater fish. In ouranalysis, the patin concentration is different in 1:10 dilution and 1:20 dilutioncompared to siakap may due to error during carrying out the experiment.

f) Another method that can be used for determination of protein content in foodsample is Lowry method. Lowry reaction consists of the Biuret reaction followedby the reduction under alkaline condition using a reagent. Copper ions facilitatethe reduction process.(Folin, 1927) The difference between BCA and Lowry isthat in Lowry, the method relies on the color development from the Biuretreaction and from the reduction of an arsenomolybdate reagent (the Folin-Ciocalteau reagent) by the tyrosine and tryptophan residue in the treated protein

while in BCA bichinchoninic acid is used. The colour in Lowry method is bluecompared to BCA which is purple and the absorbtion in Lowry method is around750nm compared to BCA which is 560nm (Krohn, 2002)


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ConclusionThe protein concentration of patin fish and siakap fish were obtained. In sample 1:10,the concentration of patin fish is higher compared to siakap fish, while in sample 1:20,the concentration of siakap fish is higher than patin fish.

References

Suzanne S.N. ,2010Analysis Laboratory Manual, Food Science Texts Series, pp315;The Ohio State University : USA

Sigma-Aldrich, 2013 Protein Determination by the Bicinchoninic Acid (BCA) Method[Online] Available :http://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer/protein-determination-by-the-bicinchoninic-acid-bca-method.html

Thermo Scientific, 2013 Chemistry of Protein Assays [Online] Available : http://www.piercenet.com/browse.cfm?fldID=876562B0-5056-8A76-4E0C-B764EAB3A339

Krohn, R.I. (2002). The Colorimetric Detection and Quantitation of Total Protein, CurrentProtocols in Cell Biology , A.3H.1-A.3H.28, John Wiley & Sons, Inc

Folin, O. and Ciocalteau, P. (1927) Journal of Biology and Chem. 73:627
http://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer/protein-determination-by-the-bicinchoninic-acid-bca-method.htmlhttp://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer/protein-determination-by-the-bicinchoninic-acid-bca-method.htmlhttp://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer/protein-determination-by-the-bicinchoninic-acid-bca-method.htmlhttp://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer/protein-determination-by-the-bicinchoninic-acid-bca-method.htmlhttp://www.piercenet.com/browse.cfm?fldID=876562B0-5056-8A76-4E0C-B764EAB3A339http://www.piercenet.com/browse.cfm?fldID=876562B0-5056-8A76-4E0C-B764EAB3A339http://www.piercenet.com/browse.cfm?fldID=876562B0-5056-8A76-4E0C-B764EAB3A339http://www.piercenet.com/browse.cfm?fldID=876562B0-5056-8A76-4E0C-B764EAB3A339http://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer/protein-determination-by-the-bicinchoninic-acid-bca-method.htmlhttp://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer/protein-determination-by-the-bicinchoninic-acid-bca-method.html

lab exercise 2 - fish muscle protein (autosaved).docx

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