Laboratory manual of biochemistry: methods and techniques/ R.S Sengar

Unit1. General Laboratory Principles 1
Unit 2. Carbohydrates 11
Experiment - 1 : Analysis of Carbohydrates 14
Experiment - 2 : Estimation of Reducing Sugar by
Dinitrosali cylate (DNS) and Plotting
Standard Curve by Dextrose 23
Experiment - 3 : Estimation of Sugars by Orcinol Method 25
Experiment - 4 : Estimation of Reducing Sugars by
Nelson-Somogyi's Method 27
Experiment - 5 : Estimation Of total Soluble Sugars by
Anthrone Method 30
Experiment - 6 : Estimation of total Soluble Sugars By
Phenol - Sulphuric Acid Method 32
Experiment - 7 : Estimation of Non-Reducing Sugars 34
Experiment - 8 : Estimation of Starch by Anthrone Reagent ... 36
Experiment - 9 : Determination of Amylose 38
Experiment - 10 : Estimation of Maltose from any Fruit
Juice 40
Experiment - 11 : Estimation of Cellulose from Fresh Plants .... 42
Experiment - 12 : Estimation of Hemicellulose 44
Experiment - 13 : Estimation of Crude Fibre 46
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Contents/xii
Unit 3. Amino Acids and Proteins 49
Experiment - 1 : Identification of N-terminal Amino Acid of
a Protein using Dansyl Chloride Method 51
Experiment - 2 : Isolation of Milk Protein (Casein) from the
Milk 54
Experiment - 3 : Estimation of Total Protein from Plant Fresh
Tissue 56
Experiment - 4 : Estimation of Total Amino Acid from Plant
Fresh Tissue 58
Experiment - 5 : Identify the Amino Acid present in the
given sample by Qualitative Method 61
Experiment - 6 : Estimate the Concentration of Amino Acids
in given sample by Ninhydrin Method 65
Experiment - 7 :
Estimate the Concentration of Protein in
given sample by Biuret Method 68
Experiment - 8 :
Estimate the Concentration of Protein in
given sample by Folin-Low^ry's Method 71
Experiment - 9 : Estimate the Concentration of Protein in
given sample by Bradford's Method 74
Experiment - 10 : Estimation of Amino Acid by
Spectrophotometric Method 77
Experiment - 11 : Native Disc-Gel Electrophoresis of
Proteins 79
Experiment - 12 : Separation of Proteins by SDS-PAGE 82
Experiment - 13 : SDS- Polyacrylamide slab Gel
Electrophoresis of Proteins under Reducing
Conditions (SDS-PAGE) 86
Experiment - 14 : Estimation of total Nitrogen by Micro -
Kjeldahl Method 92
Unit 4. Lipids 97
Experiment - 1 : Extraction and Gravimetric Estimation
of lipid from Ground Nut by Soxhlet
Method 98
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Experiment - 2 : Estimate the Lipid Present in given
sample by Sulfo-Phospho Vanillin Method 100
Experiment - 3 : Determination of Acid-Value of Fats and
Oils 102
Experiment - 4 : Determination of Saponification Value of
Fat and Oil 105
Experiment - 5 : Estimation of Free Fatty Acids 108
Experiment - 6 : Determination of Iodine Value of Oil 110
Experiment - 7 : Determination of Peroxide Value of Oil 112
Experiment - 8 : Estimation of Volatile Acids 114
Experiment - 9 : Identification and Quantification of Fatty
Acids 116
Experiment -10 : Steam Distillation and Isolation of Essential
Oils 118
Unit 5. Nucleic Acids 12i
Experiment - 1 : The Determination of the Phosphorus
content of a Nucleic Acid 124
Experiment - 2: Estimation of DNA by Diphenylamine
Experiment - 3
Experiment - 4
Experiment - 5
(DPA) Method 125
Determination of RNA by Orcinol Method ... 127
To Perform Restriction Digestion of DNA .. 129
To Perform Ligation of EcoRI Digest using
T4 DNA Ligase 130
Experiment 6:
Elution of Plant DNA Fragment from
Agarose Gel 132
Experiment - 7: Isolation of total RNA 134
Experiment - 8 : To Perform Southern Hybridization 136
Experiment - 9 : To Perform
PGR Amplification of Specific
Target Sequence from Genomic DNA 141
Experiment - 10 : To Isolate the total Genomic DNA from
Plant Source 143
Contents/xiv
Unit 6. Enzymes
Experiment - 1 : Determination of Catalytic Power of Acid
Phosphatase
Experiment - 2: To Obtain the Progress Curve During the
Hydrolysis of p-Nitrophenyl Phosphate by
Serum AlkalinePhosphatase (Ortliophospilioric
Monoester Phospliohx/iirolasc, 3.1.3 152
Experiment - 3: Variation in Serum Alkaline Phosphatase
Activity with Enzyme Concentration 155
Experiment - 4: The Determination of the Michaelis
Constant for the Digestion of Casein by
Trypsin (3.4.4.4.) ^60
Experiment - 5 : Yeast Isocitrate Dehydrogenase: An
Allosteric Enzyme ^65
Experiment - 6 : Nicotinamide Adenine Dinucleotide as
the Coenzyme for Lactate Dehydrogenase
(L-lactate: NAD Oxidoreductase, 1.1.1.27) 168
Experiment - 7: The Effect ofInhibitors on Ox Heart
Lactate Dehydrogenase 173
Experiment - 8 : Determination of Nitrate Reductase
Activity ^78
Experiment - 9 : Demonstrate the Diastase Activity in
Germinating Seeds of Barley 181
Experiment - 10 : The Determination of the 'Optimum
Terhperature' of a-amylase 184
Experiment - 11 :The
Effect of Temperature on the Stability
of a-amylase ^86
Experiment - 12 :The pH Optimum of an Enzyme 189
Experiment - 13 : Determination of Enzyme Glutamate
Dehydrogenase Activity (GDH) from Plant
Leaves/Shoot/Root 190
Experiment - 14 : Determination of Enzyme Glutamine
Synthetase Activity (GS) from Plant Tissue ..193
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Experiment - 15 : Determination of Enzyme Glutamate
Synthase Activity (GOGAT) from Plant
Tissue 195
Experiment -16 : Estimate the Amylase Activity in the given
Sample by Calculating Units
(U) of Enzyme... 198
Experiment - 17: investigate the Effect of Temperature
on Amylase Activity and find out the
Temperature Optima for Amylase 201
Experiment-18 : Estimation of Catalase Enzyme from Plants....203
Experiment - 19 : Estimation of Peroxidase Enzyme from
Fresh Leaves 205
Experiment - 20 : Estimation of Superoxide Dismutase Enzyme
from Plant Tissue 207
Experiment - 21: Estimation of Ascorbate Peroxidase
Enzyme from Plants 211
Experiment - 22: Estimation of Ascorbate Content
Determination from Fresh Leaves 213
Unit 7. Vitamins 217
Experiment - 1 : Estimation of Ascorbic Acid (Vitamin C) by
Volumetric Method 219
Experiment - 2 : Estimation of Ascorbic acid (Vitamin C)
by Colorimetric Method 221
Estimation of Vitamin A 224
Estimation of Vitamin D2 and D3 227
Estimation of Tocopherol (Vitamin E) 229
Estimation of Pyridoxine (B^) 232
Estimation of Thiamin 235
Estimation of Riboflavin 238
Unit 8. Pigments 243
Experiment - 1 : Estimation of
Total Carotenoids from Plant
Tissue 246
Coiitents xvi
Experiment - 2: Estimation of Chloropliyll from Plant
Tissue 249
Experiment - 3: To Separate Anthocynins by Paper and
/or Thin layer Chromatography
Experiment - 4; To Separate and Characterize the
Anthocyanin Pigments
Experiment - 5: Colour Development in Apples under the
Influence of Quality of Light
Experiment - 6: To Separate and Identify the Lycopene
Present in Plant Material
Experiment - 7: To Isolate the Chloroplast Pigments on the
Basis of their Differential Solubility in
Various Organic Solvents and theii
Absorption Properties using Paper
Chromatography
Experiment -8: Quantitative Determinadon Study of
Absorption Spectrum of Chloi ophy11s
Extracted from the Leaves of Higher Plants....266
Experiment - 9: Isolation of Active Chloroplasts fromExperiment observation of Hill Reaction
Activity of Chloroplasts
Experiment -10 :To Extract the Curcumin Pigment 270
Unit 9. Phenolics
Experiment -1: To Estimate Total Phenol Content in Plant ...275
Experiment - 2: To Estimate the Chlorogenic Acid 277
Experiment - 3: To Estimate the Tannins by Folin-Denis and
Vanillin Hydroclonde Methods
Experiment - 4: To Estimate the Lignins
Experiment - 5; To Estimate the Capsaicin by ColonmetricExperiment gpectrophotometric Methods 285

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Unit10. Anti Nutritional Factors 289
Experiment - 1 : Estimation of Trypsin Inhibitor 291
Experiment - 2 : Determination of Cyanogens 293
Experiment - 3 : Estimation of Phytic Acid in Food Grains .... 295
Experiment - 4 : To Study the Cysteine Proteinase Inhibitor
Assay 297
Experiment - 5: To Study the Antifungal Activity of
Cystatin Present in the Given Sample 299
Unit 11. Plant Hormones
Experiment - 1 : Identify the Indoleacetic Acid Present in the
Given Sample 305
Experiment - 2 : Estimation of Ethylene in the Plant