Basic Techniques inBiochemistry andMolecular Biology

Basic Techniques inBiochemistry andMolecular Biology

R.K. SharmaChairman & Head

Reproductive Biology Lab., department of ZoologyKurukshetra University, Kurukshetra

Haryana

S.P.S. SanghaDepartment of Biochemistry

Guru Angad Dev University of Veterinary and Animal SciencesLudhiana, Punjab

I.K. International Publishers Pvt. Ltd.NEW DELHI • BANGALORE

Published byI.K. International Publishers Pvt. Ltd.S-25, Green Park ExtensionUphaar Cinema MarketNew Delhi–110 016 (India)E-mail: ik_in@vsnl.net

ISBN 978-81-907462-

© 2009 Copyright Reserved

All rights reserved. No part of this book may be reproduced or used in any form,electronic or mechanical, including photocopying, recording, or by any informa-tion storage and retrieval system, without written permission from the publisher.

Published by Krishan Makhijani for I.K. International Pvt. Ltd., S-25, GreenPark Extension, Uphaar Cinema Market, New Delhi–110 016. Printed by RekhaPrinters Pvt. Ltd., Okhla Industrial Area, Phase II, New Delhi–110 020.

Dedicated our Mentors

Prof. P.L. Duda&

Prof. S.S. Guraya

The exponential growth in Life Sciences is largely due to theapplications of Biochemistry and Molecular Biology. Thedevelopment of methodologies and procedures have advanced theacademic dimensions and their applications have improved thegoods and services in food, drinks and pharmaceutical industries.Quality control, clinical diagnostics, public health and pollutionabetment sectors are largely dependent on Biochemistry andMolecular Biology techniques.

In the era of multidisciplinary approach, the basic techniques inBiochemistry and Molecular Biology are much needed by thestudents of Botany, Zoology, Microbiology, Biotechnology,Fisheries, Veterinary, Pharmacology, Physiology, Medicine,Genetics, Agriculture and allied subjects both at undergraduate andpostgraduate levels. It is in this context that the present bookwritten by hardworking teachers, namely, Dr. Rajnesh K. Sharmaand Dr. S.P.S. Sangha is most timely and is a valuable contribution.

The book is divided in 15 chapters covering more than 135experimental protocols ranging from qualitative tests to PCR andblotting techniques. All the experiments are clearly designed so thatthe beginner can perform. I am sure that this book will be of great

Foreword

help to undergraduate and postgraduate students, research scholarsand teachers.

Dr. Hari SinghProfessor Emeritus

Department of BiochemistryKurukshetra University

Kurukshetra

viii Foreword

The concept of and approach to Biology has undergone anunprecedented growth in all directions. To explain life andfunctioning of a living organism, the molecular bases are beingexplored on war footing. Fundamentals of biochemistry andmolecular biology is an important component of all disciplines ofBiology such as Agriculture, Botany, Microbiology, Biotechnology,Aquatic Biology, Veterinary Sciences, Pharmaceutical Science,Zoology, Immunology, Genetics, Physiology and Medicine atundergraduate and postgraduate levels.

In the era of multidisciplinary approach both students findteachers and difficulties in devising and executing experimentsdealing with Biochemistry and Molecular Biology. The presentbook is so designed that a beginner can easily perform theseexperiments and pursue further studies in this important area.

This book includes 15 chapters covering basic topics like pHand buffers, spectrophotometry, chromatogrphy, carbohydrates,lipids, proteins, electrophoresis, enzyme immunology, vitamins andpigments, metabolites and molecular biology. It includes a widerange of experiments from preparations of culture media to PCR,Southern and Western blotting. All experiments have beenmeticulously designed and special care has been taken to the safetyin laboratory and precautions are given wheresoever required.

Preface

The authors are thankful to Dr. G.D. Sharma, Centre ofBiotechnology (CSIR), New Delhi for his generous help. Specialthanks are due to Dr. (Mrs) Anita Bhatnagar, Dr. Baljeet Singh(Dept. of Microbiology) and Mr. Jitender Kumar for theircontributions and critical analysis. The cooperation rendered byfellow colleagues in Department of Zoology at KurukshetraUniversity, Kurukshetra and Department of Biochemistry, GuruAngad Dev University of Veterinary and Animal Sciences,Ludhiana, is duly acknowledged. Lastly Dr. R.K. Sharma isthankful to his wife Dr. (Mrs) Manju Sharma and son Sushank forallowing him to encroach upon their time, and Dr. S.P.S. Sangha isgrateful to his mother, wife Dr. (Mrs) G.K. Sangha and brother andsister-in-low for the nice cooperation. Prof. Sangha records hisgratitude to Purneet, Karn Amar & Alam.

R.K. SharmaS.P.S. Sangha

x Preface

Chapter 1: General Introducton 1

Laboratory Safety 1Units and Quantities 3Solutions of Varied Strengths 4Practical Record 6

Chapter 2: pH and Buffers 8

Acids and Bases 8Hydrogen Ion Concentration and pH 9Measurement of pH 9Buffers 10

2.1 Determination of pH of a solution using 11common chemical indicators

2.2 Titration of gastric juice with 0.1 M of KOH 122.3 Titration of weak acid with a strong base to plot 13

the changes in pH in titration curve using a pH meter2.4 The determination of pKa values of a

dicarboxylic acid 13

Chapter 3: Spectrophotometry 15

The Beer – Lambert Law 15General Instruction for the use of spectrophotometers 18

Contents

xii Contents

3.1 Demonstration of absorbance curves of bromophenolblue and potassium permanganate 22

3.2 The colorimetric demostration of Beer-Lambert law 243.3 Estimation of creatinine and verification of

Beer-Lambert law 243.4 Demonstration of nucleic acids (DNA and RNA)

by spectrophotometric method 26

Chapter 4: Centrifugation and Cell Fractionation 28

4.1 To prepare sub-cellular fractions of the liver cells 314.2 To isolate mitochondria from rat liver 324.3 To isolate lysosomes from rat liver by density

gradient centrifugation 334.4 To fractionate cellular components of spinach leaves 344.5 Preparation of protoplasts from spinach leaves 354.6 Isolation of mitochondria from spinach leaves 364.7 Isolation of chloroplasts from spinach leaves 38

Chapter 5: Chromatography 39

Column chromatography 40Low-Pressure Column Chromatography 42Preparation of the Column 43

5.1 To determine the molecular weight of a protein bygel filtration chromatography 47

5.2 The separation of two compounds on the basis oftheir molecular weights by column chromatography 48

5.3 Demonstration of desalting of a protein solutionusing column chromatography 49

Adsorption Chromatography 505.4 Demonstration of adsorption chromatography in

separation of leaf pigments using a piece of chalk 515.5 Preparation of an adsorption chromatographic column

and demonstration of separation leaf pigments 52

Contents xiii

Ion Exchange Chromatography 535.6 The uptake of potassium chloride by a cation

exchange resin 535.7 Demonstration of the separation of amino acids by

ion exchange chromatography 54Paper Chromatography 56

5.8 Separation and identification of the amino acids fromthe given sample by paper chromatography 56

Thin Layer Chromatography 60

Chapter 6: Carbohydrates 66

6.1 General qualitative tests for carbohydrates 676.2 Identification of specific carbohydrate present in

the solution provided 686.3 To hydrolyse polysaccharides 746.4 Estimation of Carbohydrates by anthrone method 756.5 Determination of reducing sugar by Nelson

Somogyi method 776.6 Estimation of total carbohydrates by phenol sulphuric

acid method 786.7 Estimation of reducing sugars by dinitrosalicylic

acid method 796.8 Estimation of starch by anthrone method 816.9 Determination of fructose content of the sample 82

6.10 Estimation of inulin 836.11 Determination of amylose content 836.12 Estimation of cellulose 856.13 Estimation of hemicellulose 866.14 Estimation of acid detergent fibre 876.15 Estimation of pectin by gravimetric method 886.16 Estimation of crude fibre 90

Chapter 7: Lipids 92

7.1 Perform qualitative tests for lipids 927.2 Estimation of lipid content of the oilseeds. 94

xiv Contents

7.3 Extraction and estimation of total lipids from animaltissue by Folch method 95

7.4 Determination of the acid value of the fat sample 967.5 Determination of saponification value of a fat sample 977.6 Determination of iodine number of the given fat 987.7 Estimation of cholesterol 1007.8 Estimation of free fatty acids 1017.9 Estimation of triglycerides content from the

lipid sample 1027.10 Estimation of phospholipids 1037.11 Determination of glycolipids 104

Chapter 8: Amino Acids and Proteins 105

8.1 Qualitative test of proteins and amino acids 1068.2 Demonstration of denaturation of proteins 1078.3 General tests for amino acids 1098.4 Estimation of proteins by micro-Kjeldhal method. 1148.5 Extraction and estimation of amino acids from

tissue/foodstuffs 1168.6 Estimation of total amino acids 1198.7 Estimate includes only a-amino acids 1208.8 Estimation of tryptophan 1228.9 Estimation of methionine 123

8.10 Estimation of lysine from cereals grains 1248.11 Estimation of proteins by Biuret method 1278.12 Estimation of proteins by Folin-Lowry method 1288.13 Protein estimation by Bradford method 1298.14 Estimation of protein by Warburg-Christian method

(UV–method) 130

Chapter 9: Nucleic Acids 132

9.1 To extract nucleic acids from plant tissues 1329.2 Preparation of extract for the estimation of DNA and

RNA content from animal tissues 133

Contents xv

9.3 Determination DNA content by diphenylaminemethod 134

9.4 Estimation of RNA by orcinol method 1359.5 Determination of DNA and RNA contents by

UV spectrophotometric method 1369.6 Determination of individual bases in DNA 1379.7 Determine the melting temperatureTm and (G + C)%

in the given DNA sample 1399.8 Isolation of DNA from blood sample 140

Chapter 10: Electrophoresis 142

10.1 Separation of protein fractions by SDS-PAGE 14210.2 Separation of proteins by agarose gel electrophoresis 14710.3 Separation of proteins by starch gel electrophoresis 14810.4 Western blotting of proteins from

SDS-polyacrylamide gel 15010.5 Separation of DNA fragments by agarose gel

electrophoresis 152

Chapter 11: Immunology 154

11.1 Demonstration of agar gel precipitation test (AGPT) 15411.2 Single radial immunodiffusion (SRID) 15711.3 Demonstration of enzyme linked immunosorbent

assay (ELISA) or enzyme immuno assay (EIA) 15711.4 Indirect DOT ELISA 15911.5 To detect various antigen-antibody systems in the

given sample by agrose immunoelectrophoresis 160

Chapter 12 : Enzymes 163

12.1 Determination of alkaline phosphatase from serum 16312.2 Determination of acid phosphatase from serum. 16512.3 Estimation of serum glutamate pyruvate transaminase

(GPT) alanine transaminase activity (ALT) 16612.4 Estimation of serum lactate dehydrogenase activity 16912.5 Assay of superoxide dismutase (SOD) 171

xvi Contents

12.6 Assay of glutathione peroxidase (GPX) 17212.7 Assay of glucose-6-phosphate dehydrogense (G6PD) 17412.8 Assay of 5¢ nucleotidase activity 17512.9 Assay of lactic dehydrogenase from the tissue sample 176

12.10 Determination of lactic dehydrogenase kinetics 17712.11 Estimation of sorbitall dehydrogenase 17812.12 Estimation of aryl sulphatase 17912.13 Estimation of b-galactosidase 18012.14 Estimation of b-glucoronidase 18112.15 Estimation of leucine aminopeptidase (LAP) 183

Chapter 13: Metabolites 185

13.1 Estimation of pyruvic acid 18513.2 Estimation of lactic acid 18713.3 Estimation of blood glucose 19013.4 Estimation of serum urea 19313.5 Estimation of serum creatinine 19513.6 Estimation of uric acid from serum 19613.7 Estimation of total proteins in plasma 19813.8 Estimation of serum cholesterol 19913.9 Determination of serum triacylglycerols 201

13.10 Estimation of pentasuria by Bial’s reaction 20213.11 Estimation of fructosuria by Selivanoof’s method 20313.12 Determination of phosphorus from animal tissue 20313.13 Determination of plasma calcium 20413.14 Determination of lipid peroxide oxidation rate in

tissue by malonic dialdehyde 205

Chapter 14: Vitamins, Hormones and Pigments 208

14.1 Estimation of vitamin E 20814.2 Estimation of vitamin C 20914.3 Estimation of b-carotene 21014.4 Estimation of adrenalin by ferric chloride method 211

Basic Techniques In Biochemistry AndMolecular Biology

Publisher : IK International ISBN : 9788190746267Author : R.K. Sharma,S.P.S. Sangha

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