Biochemical Techniques

by Robyt, John F.; White, Bernard J.

• ISBN13: 9780881335569
• ISBN10: 0881335568
• Edition: Reprint
• Format: Hardcover
• Copyright: 1990-01-01
• Publisher: Waveland Pr Inc

Table of Contents

Analyzing and Reporting Experimental Data	1	(20)
Recording and Handling Experimental Data	1	(4)
Significant Figures	1	(2)
Scientific Notation	3	(1)
Units	3	(2)
Analysis of Experimental Data	5	(4)
Error Analysis and the Estimation of Precision	5	(3)
Errors and the Rejection of Data	8	(1)
Calculating the Uncertainty of the Result of Mathematical Operations	8	(1)
The Relation Between Precision and the Number of Determinations	9	(1)
Tables and Graphs	9	(8)
Tables	9	(1)
The Use of Powers of 10 in Tables and Figures	10	(1)
Rules for Making Graphs	11	(2)
Linearization	13 (	(1)
Numerical Curve Fitting: The Method of Least Squares	14	(2)
Weighting Factors in Linear Least Squares	16	(1)
Calculating the Area Under a Curve	16	(1)
Controls and Blanks	17	(1)
General Rules for Nomenclature	17	(1)
The Laboratory or Research Report	18	(1)
Literature Cited	19	(1)
References for Further Study	19	(2)
Preparation and Properties of Solutions	21	(19)
Methods of Measurement	21	(1)
Volumetric Glassware	21	(5)
Use of All Volumetric Glassware	26	(1)
Cleaning	26	(1)
Reading the Meniscus	27	(1)
Calibration	27	(1)
The Analytical Balance	27	(2)
Expression of Concentration and Dilution	29	(1)
Making Dilutions	30	(2)
Standard Solutions	32	(1)
pH and Buffers	32	(5)
pH	32	(2)
Buffers	34	(1)
Buffer Capacity and Ionic Strength	35	(2)
pH Measurement	37	(2)
pH Meters	38	(1)
Making the pH Measurement	39	(1)
Literature Cited	39	(1)
References for Further Study	39	(1)
Spectroscopic Methods	40	(33)
Electromagnetic Radiation and Spectra	40	(4)
Quantitative Aspects of Light Absorption	44	(7)
The Lambert-Beer Law	44	(1)
Definition of Extinction Coefficients	45	(1)
Determining an Ultraviolet or Visible Spectrum of a Substance and the Wavelength of Maximum Absorption	46	(1)
Determining an Extinction Coefficient and concentration	46	(3)
Determining the Concentration of Two Substances in a Mixture	49	(1)
Difference Spectroscopy	50	(1)
Ultraviolet and Visible Spectrometry	51	(1)
Instrumentation	51	(1)
Applications of Ultraviolet and Visible Spectroscopy	52	(1)
Fluorescence Photometry	52	(4)
Fluorescence	52	(1)
Instrumentation	53	(3)
Infrared Spectroscopy	56	(4)
Infrared Spectrophotometry	56	(2)
Infrared Spectrophotometers	58	(1)
Sample Handling for Infrared Spectroscopy	59	(1)
Applications of Infrared Spectroscopy	59	(1)
Nuclear Magnetic Resonance Spectrometry	60	(11)
Proton NMR Spectrometry	63	(3)
Carbon-13 NMR Spectrometry	66	(1)
NMR Spectra of Other Nuclei	67	(3)
Uses of NMR in Biochemistry	70	(1)
Literature Cited	71	(1)
References for Further Study	72	(1)
Chromatographic Techniques	73	(1)
PART I. TYPES OF CHROMATOGRAPHY AND CHROMATOGRAPHIC TECHNIQUES	73	(31)
Adsorption Chromatography	74	(5)
Ion-Exchange Chromatography	79	(2)
Liquid-Liquid Partition Chromatography	81	(1)
Paper Chromatography	82	(5)
Thin-Layer Chromatography	87	(1)
Gel Permeation Chromatography	88	(7)
Preparation of Gels and Columns	94	(1)
Definitions Used in Gel Permeation Chromatography	95	(1)
Affinity Chromatography	95	(4)
The Inert Support and the Ligand	96	(1)
Attachment of the Ligand to the Solid Support	96	(2)
Hydrophobic Chromatography	98	(1)
Gas-Liquid Chromatography	99	(2)
High Performance Liquid Chromatography	101	(3)
PART II. SPECIFIC APPLICATIONS OF CHROMATOGRAPHY	104	(280)
Carbohydrates	104	(7)
Adsorption Chromatography of Carbohydrates	104	(1)
Ion-Exchange Chromatography of Carbohydrates	105	(1)
Paper Chromatography of Carbohydrates	106	(1)
Thin-Layer Chromatography of Carbohydrates	107	(2)
Gel Permeation Chromatography of Carbohydrates	109	(1)
Affinity Chromatography of Carbohydrates	109	(1)
Gas-Liquid Chromatography of Carbohydrates	109	(2)
High Performance Liquid Chromatography of Carbohydrates	111	(1)
Amino Acids	111	(4)
Ion-Exchange Chromatography of Amino Acids	111	(1)
Thin-Layer Chromatography of Amino Acids	112	(2)
High Performance Liquid Chromatography of Amino Acids	114	(1)
Proteins	115	(3)
Adsorption Chromatography of Proteins	115	(1)
Ion-Exchange Chromatography of Proteins	116	(1)
Gel Permeation Chromatography of Proteins	116	(2)
High Performance Liquid Chromatography of Proteins	118	(1)
Nucleotides and Nucleic Acids	118	(3)
Ion-Exchange Chromatography of Nucleic Acids	118	(1)
Gel Permeation of Nucleic Acids	119	(1)
Affinity Chromatography of Nucleic Acids	119	(1)
High Performance Liquid Chromatography of Nucleic Acids	120	(1)
Lipids	121	(3)
Adsorption Chromatography of Lipids	121	(1)
Ion-Exchange Chromatography of Lipids	121	(1)
Paper Chromatography of Lipids	121	(1)
Thin-Layer Chromatography of Lipids	121	(2)
Gas-Liquid Chromatography of Lipids	123	(1)
High Performance Liquid Chromatography of Lipids	123	(1)
Literature Cited	124	(4)
References for Further Study	128	(1)
Electrophoretic Techniques	129	(29)
Theory	129	(1)
Types of Electrophoresis	130	(20)
Paper and Cellulose Acetate Electrophoresis	130	(3)
Thin-Layer Electrophoresis	133	(1)
Gel Electrophoresis	133	(13)
Immunoelectrophoresis	146	(2)
Isoelectric Focusing	148	(2)
Specific Applications	150	(6)
Proteins and Peptides	150	(1)
Nucleic Acids and Oligonucleotides	151	(5)
Lipoprotein Separation	156	(1)
Literature Cited	156	(1)
References for Further Study	157	(1)
Theory, Measurement, and Use of Radioisotopes	158	(55)
Nature of Atoms, Isotopes, and Radioisotopes	158	(1)
Types of Radioactive Decay	159	(3)
Decay by Negative-Beta (Electron) Emission	159	(1)
Decay by Positive-Beta (Positron) Emission	160	(1)
Decay by Electron Capture	160	(1)
Decay by &gamma:-Radiation	161	(1)
Decay by &alph;-Particle Emission	161	(1)
Occurrence and Artificial Production of Radioactive Isotopes	162	(1)
Properties of Radioactive Emissions	163	(8)
Energy of β-Particle Emissions	163	(1)
Interactions of β-Particles with Their Environment	164	(1)
Gamma Radiation and Its Interaction with Matter	165	(1)
Kinetics of Radioactive Decay	166	(5)
Units Used in Radioactive Measurements	171	(1)
The Electron Volt	171	(1)
The Curie: The Unit of Radioactive Disintegration	171	(1)
Special Techniques and Safety in Handling Radioisotopes	172	(1)
Statistics of Counting Radioisotopes	173	(3)
Measurement of Radioactivity by Gas Ionization	176	(4)
Measurement of Radioactivity by Scintillation Counting	180	(10)
Mechanism of Liquid Scintillation Counting	180	(1)
Pulse Height Analysis and the Beta Energy Spectrum	181	(1)
Determining the Activity of Each Isotope in a Dual-Labeled Mixture	182	(2)
Cocktail Composition and Sample Preparation	184	(2)
Quenching and Quench Correction	186	(4)
Solid Scintillation Counting of γ-Radiation	190	(1)
Detection by Autoradiography	191	(2)
Methods of Labeling Biochemical Compounds	193	(4)
Types of Radiochemical Labeling	197	(2)
Applications in the Use of Radioisotopes	199	(10)
The Quantitative Determination of Compounds by Use of Radioisotopes	200	(1)
The Quantitative Determination of a Compound by Isotope Dilution Analysis	201	(3)
The Determination of the Mole Fractions of Several Compounds Formed from One Labeled Precursor	204	(1)
Pulse and Chase Techniques with Radioisotopes	205	(1)
The Use of Radioactive Tracers in the Study of Enzyme Reaction Mechanisms and Metabolic Pathways	206	(1)
Radioimmunoassay	207	(2)
Designing a Radioisotope Experiment	209	(3)
Literature Cited	212	(1)
References for Further Study	212	(1)
Qualitative and Quantitative Methods for Determining Biological Molecules	213	(40)
Carbohydrates	213	(14)
Qualitative Tests for Different Types of Carbohydrates	213	(4)
The Quantitative Determination of Carbohydrates	217	(10)
Amino Acids, Peptides, and Proteins	227	(12)
Qualitative Tests for Amino Acids and Proteins	227	(2)
The Quantitative Determination of Amino Acids	229	(2)
The Quantitative Determination of Proteins	231	(8)
Nucleotides and Nucleic Acids	239	(4)
The Qualitative Determination of RNA and DNA	240	(1)
The Quantitative Determination of Nucleotides and Nucleic Acids	241	(2)
Lipids and Steroids	243	(4)
Qualitative Tests for Lipids on Thin-Layer Chromatograms	243	(2)
Qualitative Tests for Steroids	245	(1)
The Quantitative Determination of Lipids	246	(1)
Miscellaneous	247	(3)
The Determination of Inorganic Phosphate: Modified Fiske--Subbarow Method	247	(1)
Specific Binding Assays	247	(3)
Literature Cited	250	(2)
References for Further Study	252	(1)
Biological Preparations	253	(38)
The Starting Material	253	(1)
Cell Lysis and Extraction	254	(2)
Special Techniques in Preparing Biological Materials	256	(9)
Centrifugation	256	(3)
Density Gradient Centrifugation	259	(1)
Filtration	259	(2)
Concentration of Solutions	261	(2)
Dialysis	263	(1)
Growth of Bacteria for the Production of Biochemical Compounds	264	(1)
Subcellular Fractionation of Cell Components and Organelles	265	(2)
Preparation of Liver Mitochondria	265	(1)
Preparation of Chlorophasts	266	(1)
Isolation of Liver Nuclei	266	(1)
Preparation of Escherichia Coli Ribosomes	267	(1)
Purification of Proteins	267	(8)
Separations Based on Solubility	268	(2)
Separation by Chromatography and Electrophoresis	270	(1)
Crystallization of Proteins	270	(1)
Isolation and Purification of Specific Proteins	271	(4)
Preparation of Carbohydrates	275	(3)
General Separation and Purification Methodology	275	(1)
Specific Methods for Preparing Different Carbohydrates	275	(3)
Preparation of Nucleic Acids	278	(6)
General Separation and Purification Methodology	278	(2)
Specific Methods for Preparing Different Nucleic Acids	280	(4)
Preparation of Lipids	284	(5)
General Separation and Purification Methodology	284	(1)
General Extraction and Purification Procedures	284	(1)
Specific Methods for Preparing Lipids	285	(4)
Literature Cited	289	(1)
References for Further Study	290	(1)
Enzymology	291	(30)
Kinetics and Theory of Enzyme Action	292	(3)
Determining the Initial Velocity vi	295	(1)
Enzyme Reactions as a Function of pH	296	(1)
Enzyme Reactions as a Function of Temperature	297	(1)
Determining the Activity of an Enzyme	298	(6)
Determining the Michaelis Constant Km, the Maximum Velocity Vm and the Turnover Number k2 of an Enzyme	304	(2)
Inhibition of Enzymes	306	(10)
Competitive Inhibition	307	(3)
Noncompetitive Inhibition	310	(1)
Mixed Inhibition	310	(3)
Uncompetitive Inhibition	313	(3)
Allosteric Enzymes That Do Not Follow Michaelis-Menton Kinetics	316	(2)
Specificity of Enzymes	318	(1)
Literature Cited	318	(2)
References for Further Study	320	(1)
Structural Analysis of Biological Molecules	321	(63)
Determining the Structure of Oligosaccharides and Polysaccharides	322	(16)
Determining the Monosaccharide Composition	323	(1)
Determining the Structure of Oligosaccharides	324	(10)
Determining the Structure of Polysaccharides	334	(4)
Determining the Primary Structure of Peptides and Proteins	338	(14)
The Formation and Separation of Polypeptide Chains	339	(1)
Establishing the Purity of the Protein	340	(1)
Determining the Amino Acid Composition	340	(1)
End-Group Analysis	341	(2)
Specific Methods of Peptide Bond Cleavage	343	(3)
The Separation of Peptides	346	(1)
The Sequencing of Peptides	347	(5)
Deducing the Total Sequence from Overlapping Peptides	352	(1)
Determining the Structure of Nucleic Acids	352	(20)
The Isolation, Purification, and Cloning of DNA	353	(2)
The Enzymatic Cleavage of DNA and Restriction Mapping	355	(3)
The Establishment of Purity and the Separation of DNA Strands by Electrophoresis	358	(1)
Sequencing Strategies	358	(14)
Deducing the Complete Sequence for Large Nucleic Acids	372	(1)
Determining the Structure of Lipids	372	(9)
Specific Cleavage of Lipids into Component Parts	374	(1)
Determination of Lipid Components	375	(3)
The Characterization of Phosphatidyl Choline from Egg Yolk---An Example of Lipid Analysis	378	(3)
Literature Cited	381	(3)
Appendix A: Literature Sources for Biochemical Analyses, Methods, and Preparations	384	(3)
Appendix B: Amino Acids, Abbreviations, and DNA Triplet Codes	387	(1)
Appendix C: Some Commercially Available Restriction Endonucleases and Their DNA Sequence Specificities	388	(2)
Appendix D: Table for Preparing Solutions of Different Concentrations of Ammonium Sulfate	390	(3)
Index	393