Biochemical Tests

Contents:

Colorimetry: Introduction • Principle of Colorimetric and Photometric Estimations • Beer-Lambert’s Law • Colorimetry • Methods • Absorption of Radiant Energy • Transmittance • Photoemissive Cells • Photovoltaic Cells • Double Cell Filter Photometer

Spectrophotometry: Principle • Construction of a Spectrophotometer • Source of Radiant Energy • Incandescent Lamp • Hydrogen Discharge Tube • Mercury Discharge Lamp • Globars and Nernst Glowers • Monochromators • Prisms • Grating • Slits • Cuvette or Cells • Construction of a Spectrophotometer • Relationship between Absorption in Ultraviolet and Visible Range and Molecular Structure • Determination of Absorption Spectrum and Absorption Maximum
Carbohydrates: Qualitative Tests for Sugars/Polysaccharides • Molisch Test (-Naphthol Reaction) • Moore's Test • Fehling's Test • Benedict's Test • Benedict's Reagent • Barfoed's Test • Barfoed's Reagent • Nylander's Test • Nylander's Reagent • Picric Acid Test • Seliwanoff's Test (Resorcinol Test) • Seliwanoff's Reagent • Cole's Test • Methylamine Hydrochloride Test (FEARON'S TEST) • Mucic Acid Test • Alcoholic Fermentation • Iodoform Test • Iodine Test • For Starch • Estimation of Reducing Sugars Using • Nelson–Somogyi Method • Estimation of Amount of Blood Sugar Using Nelson–Somogyi Method • Estimation of Blood Glucose by Folin-Wu Method • Estimation of Reducing Sugars by Dinitrosalicylic (DNS) Acid Method • Determination of Keto Sugars Using Roe Nicholas and Papadopoulos Method • Estimation of Total Carbohydrates Using Rex Montgomrey (phenol–HSO) Method • Estimation of Total Carbohydrates Using Anthrone Method • Estimation of Glucose Using Glucose Oxidase, Peroxidase and O-dianisidine (Trinder Test) • Isolation of Starch from Potatoes • Hydrolysis of Starch by Amylase (Saliva) • Isolation of Glycogen from Liver

Amino Acids and Proteins: Introduction • Qualitative Tests • General Composition Test for Proteins • Test for Organic Nitrogen • Test for Sulfur • Millon's Test • Millon-Nasse Test • Xanthoproteic Test • Hopkins–Cole Test • Biuret Test • Ninhydrin (Triketohydrindene Hydrate) Reaction • Sakaguchi Test • Isolation of Crystallized Albumin from Egg • Isolation of Glutenin and Gliadin from Wheat Flour • Determination of the Isoelectric Point of Casein • Isolation of Casein from Milk • Quantitative Tests for Amino Acids/Proteins • Estimation of Protein Using SpectrophotometricMethod • Estimation of Protein Using Turbidimetric Method • Estimation of Protein Using Bicinchoninic Acid (BCA) • Advantages • Estimation of Protein Using Biuret Method • Estimation of Protein Using Folin-Ciocalteau (Lowry) Method • Estimation of Tyrosine and Protein in a given Mixture Using Folin-Ciocalteau Method • Advantages and Disadvantages • Estimation of Protein Using Bradford Method • Estimation of Amino Acids Using Ninhydrin Reaction • Estimation of Amino Acid Using Sorensen Formol Titration • Estimation of Purity of an Amino Acid Sample Using Sorensen Formal Titration • Estimation of Amino Acid Nitrogen in Urine Sample Using Sorensen Formal Titration • Determination of pK of an Amino Acid

Lipids: Introduction • Qualitative Tests • Emulsification • Salting Out of Soap • Acrolein Test for Glycerol • Salkowski Test for Cholesterol • Determination of Cholesterol Content by Liebermann-Burchard Method • Baudin Test for Checking the Purity of Clarified Butter (Ghee) • Determination of Saponification Number (Koettes-Dorfers Number) of Fat • Determination of Iodine Number of Oil/Fat • Determination of Acid Value of Fat • Isolation of Cholesterol from Egg Yolk
Nucleic Acids: Introduction • Isolation of Chromosomal DNA from Bacteria • Isolation of DNA from Blood • Isolation of DNA from Plant Leaf Tissue • Estimation of DNA Concentration Using Diphe Nylamine Method • Isolation of RNA from Yeast • Isolation of Total Cellular RNA from Eukaryotes (Plant Tissue) • Estimation of RNA Concentration Using Orcinol Method • Estimation of Nucleic Acid (DNA) Using Spectrophotometric Method • Separation of DNA and RNAs • Separation of RNA, Single-stranded DNA and Double-stranded DNA using Cesium Chloride Density Gradient Centrifugation • Separation of Different Types of RNA Using Sucrose Density Gradient Centrifugation • Separation of Different Size Fragments of DNA or RNA through Electrophoresis and their Separation from Gel • Fractionation of mRNA from Total Cellular RNA • Isolation of Plasmid DNA (Miniprep) • Large-Scale Preparation of Plasmid DNA • Digestion of Plasmid DNA with Restriction Endonucleases

Vitamins: Introduction • Estimation of Vitamin C (Ascorbic Acid) Using ,-dichlorophenolindophenol (DCPIP) Dye Titration • Estimation of Ascorbic Acid in a Tissue • Determination of Ascorbic Acid Saturation in Body • Estimation of Thiamine by Thiochrome Technique

Nitrogen: Introduction • Estimation of Inorganic Nitrogen Using • Nessler's Method • Estimation of Nitrogen in the Presence of Carbon Compounds by Micro-Kjeldahl Method • (A) Digestion • (B) Distillation
Phosphorus: Introduction • Estimation of Inorganic Phosphorus Using Fiske and Subbarow Method • Estimation of Inorganic Phosphorus Using Sumner Method • Estimation of Inorganic Phosphorus Using King's Method • Estimation of Inorganic Phosphorus Using Lowry and Lopez Method • Estimation of Inorganic Phosphorus Using Berenblum and Chain Method Modified by Martin and Doty • Estimation of Organic Phosphorus

Enzymes: Introduction • Determination of the Activity of Acid Phosphatase from Cuscuta Reflexa • Determination of Activity of Acid Phosphatase from Germinating Lentil • Effect of Time on the Activity of Acid Phosphatase • Effect of Enzyme Concentration on the Activity of Acid Phosphatase • Determination of Optimum pH for Acid Phosphatase from Cuscuta Reflexa • Determination of Km and Vmax (Maximal Velocity) Values of Acid Phosphatase for Its Substrate, Glycerophosphate • Determination of Optimum Temperature for Acid Phosphatase from Cuscuta Reflexa • Determination of Inhibition Constant (Ki) of Acid Phosphatase for an Inhibitor (Fluoride) Using Dixon Plot • Fractionation of Acid Phosphatase Using Ammonium Sulfate • Determination of Activity of Starch • Phosphorylase from Banana Leaves/Indian Millet Seeds Using Colorimetry/Spectrophotometry • Determination of the Activity of Pectin Methylesterase from Vigna Sinensis Seeds Using an UV-VIS Spectrophotometer • Study the Synthesis of Amylase During Germination of Bengal Gram (Chickpea) Seeds • Enzyme Assay • Immobilization of Enzymes • Methods for Immobilization of Enzymes • I Carrier-binding Method • IA Physical Adsorption Method • Immobilization of Enzyme Starch Phosphorylase Isolated from Sorghum Leaves on Alumina by Physical Adsorption • Immobilization of Enzyme Starch Phosphorylase Isolated from Cabbage Leaves on Egg Shell by Physical Adsorption • Immobilization of Enzyme Pectin Methylesterase Isolated from Vigna Sinensis Seeds on Egg Shell by Physical Adsorption • IB Ionic Binding Method • Immobilization of Enzyme Starch Phosphorylase Isolated from Bengal Gram Seeds on DEAE-cellulose by Ionic Binding • IC Covalent Binding Method • II Cross-linking Method • Immobilization of Enzyme Starch Phosphorylase Isolated from Sorghum Leaves on Egg Shell Using Glutaraldehyde as Cross-linking Reagent • Immobilization of Enzyme Endogalacturonase Isolated from Vigna Sinensis Seeds on Sand Using Glutaraldehyde as Cross-linking Reagent • Calculation of Enzyme Activity • III Entrapping Method • Immobilization of Enzyme Pectin Methylesterase Isolated from Vigna Sinensis in Gelatin • IIIA Lattice Type • Immobilization of Enzyme Starch Phosphorylase Isolated from Sorghum Leaves in Polyacrylamide • IIIB Microencapsulated Type • Enzyme Membranes • Enzyme Tubes • Advantages of Immobilized Enzymes • Applications of Immobilized Enzymes

Milk Analysis: Introduction • Determination of Specific Gravity of Milk • Determination of pH of Milk • Determination of pH of Milk Using pH Paper • Determination of pH of Milk Using pH Meter • Isolation of Proteins from Milk • Detection of Electrolytes (Calcium and Phosphate) in Milk • Test for Calcium Ions • Test for Phosphate • Detection of Common Adulterants in Milk • Detection of Sucrose in Milk • Detection of Starch in Milk by Iodine Test • Quality Test (Presence of Microbial Cells) of Milk by Methylene Blue Dye Reduction Test • Determination of Total Solids in Milk • Determination of Fat Content of Milk

Pigments: Estimation of Chlorophyll Using Arnon Method • Estimation of -Carotene Using Spectrophotometric Method

Water Analysis: Introduction • Determination of Dissolved Oxygen in Water Sample • Determination of Chemical Oxygen Demand in Water Sample • Determination of Biochemical Oxygen Demand in Water Sample

Microbiology: Study of the Growth of Aspergillus Niger by Determination of Inorganic Phosphate and Sucrose Uptake and Measurement of Citric Acid and Mycelium Produced • Determination of Inorganic Phosphate and Sucrose Uptake and Citric Acid Production • Determination of the Amount of Citric Acid in a Plant Tissue • Growth Curve for Escherichia Coli • Study of Mutation by Ames Test

Nutrition: Proximate Analysis of Food Material • Sampling • Proximate Analysis • Vacuum Oven Method • Vacuum Desiccator • Determination of Crude Fat • Determination of Crude Proteins • Determination of Ash Content • Straight Ash Combustion Method • Glycerol • Alcohol Method • Determination of Crude Fiber • Nitrogen-free Extract • Growth Studies and Determination of Protein Efficiency Ratio • Group Feeding • Animals • Individual Feeding • Preparation of Stock Diet • Group Feeding • Nitrogen Balance Studies on Barley K • Principle • Plan of the Experiment • Material and Method • Diets • Animals • Metabolic Cages • Feeding • Weighing • Collection of Feces and Urine • Nitrogen Determination

Chromatography: Introduction • Partition Chromatography • Requirements for the Partition Chromatography • Separation of Amino Acids/Sugars by Paper Chromatography • Separation of Amino Acids Mixture by silica Gel G Thin Layer Chromatography • Separation of Plant Pigments by Thin Layer Chromatography • Gas (Gas-Liquid) Chromatography • Basic Equipment • Carrier Gas • Ion Exchange Chromatography • Determination of the Capacity of the Ion Exchanger • Isolation of Desired Enzyme by Using DEAE Cellulose Ion Exchange Chromatogrophy • (A) Regeneration of DEAE Cellulose • (B) Packing of Column with DEAE Cellulose • (C) Chromatography • Gel Filtration Chromatography • Gel Matrix • Determination of Molecular Weight of a Protein Using Sephadex G- • Whitekar Method • Method of Andrews • Affinity Chromatography • Binding of Ligand on Agarose

Electrophoresis: Introduction • Agarose Gel Electrophoresis (AGE) for Nucleic Acids • Factors Affecting Migration of DNA through Agarose Gels • Detection of Nucleic Acids • Polyacrylamide Gel Electrophoresis (PAGE) for Nucleic Acids • Polyacrylamide Gel Electrophoresis (PAGE) for Proteins • Significance of SDS-PAGE • Isoelectrofocusing • Western Blotting • Enzyme-linked Immunosorbent Assay (ELISA) • Extraction of Nucleic Acid from Agarose and Polyacrylamide Gels • Mini Agarose Gel Electrophoresis • Polyacrylamide Gel Electrophoresis for DNA Sequencing • Immunoelectrophoresis • Rocket Immunoelectrophoresis • Restriction Fragment Length Polymorphism (A Non-PCR Based Marker) • DNA Probes • Randomly Amplified Polymorphic DNA (RAPD) • Amplified Fragment Length Polymorphism (AFLP) • Southern Blotting • Northern Blotting

Centrifugation: Introduction • Differential Centrifugation • Density Gradient Centrifugation • Ultracentrifugation • Determination of Molecular Weight Using Ultracentrifugation

Polymerase Chain Reaction (PCR): Introduction • General/Traditional PCR • Materials Required • Visualizing Results • Other Important PCR • Reverse Transcription Polymerase Chain Reaction (RT-PCR) • RT-PCR Involves Two Steps • A Reverse Transcription • B. Real-time PCR

Plant Tissue Culture Experiments: Laboratory and Equipment • A Plant Tissue Culture Laboratory • B Equipment Required for a Plant Tissue Culture Laboratory • Culture Media • Plant Tissue Culture Media • Sterilization • Sterilization of the Medium • Sterilization of the Glassware •  Sterilization of the Instruments (equipment) • Sterilization of the Plant Material • Sterilization of the Transfer Area • Sterilization of the Culture Room • Single Cell Culture • Isolation of Single Cells • Culture Methods • Batch Culture • Continuous Culture • Measurement of Cell Growth in Suspension Cultures • Detection of Viability of Cells in Suspension Cultures • Cell Synchronization • In vitro Regeneration of Plants • Explants •  Callus Induction • Plant Regeneration • Organogenesis • Factors Affecting in vitro Regeneration • Micropropagation • Advantages of Micropropagation • Somatic Embryogenesis • Technique • Importance of Somatic Embryogenesis • Protoplast: Isolation, Culture and Fusion • Introduction • Selection Procedures for Fusion Products • Cybridization • Haploid Production

Appendix I • To Calibrate ml Volumetric Flask • Absolute Error or Deviation • Mean (average) Deviation • Relative Mean Deviation (RMD) • Relative Error • Relative Mean Error (RME) • Standard Deviation (S) • Variance (V) • Coefficient of Variance (CV)
Appendix II • Separation of Micromolecules and Macromolecules Using Dialysis
Appendix III • Concentration of a Sample Using Ultrafiltration
Appendix IV • Raising Polyclonal Antibodies in Rabbit
Appendix V • Ouchterlony Double Immunodiffusion Technique

About the Authors:

Dr. Anil Kumar is Full-time Professor of Biotechnology at the Devi Ahilya University, Indore, India, since 1991. He has also been heading the School of Biotechnology since then. He is a founder member of the School. He has published more than 100 full length research papers in various international and national journals of high repute. He also authored a number of books and contributed many chapters to various edited books.

Dr. Sarika Garg is a post-doctoral scientist at the University of Saskatchewan, Canada. Earlier, she worked at the Max Planck Unit for Structural and Molecular Biology, Hamburg, Germany, for more than three years. She has many research papers to her credit. She is the co-author of the book Enzymes and Enzyme Technology. She has co-authored many chapters of various edited books.

Ms. Neha Garg is a freelance writer and Entrepreneur based at Manchester, United Kingdom. Earlier she worked as Scientific Researcher at the University of Potsdam, Germany, for more than three years. She has many research papers to her credit. She is the co-author of the book Genetic Engineering and several chapters in edited books.