Holdings: Electrophoresis fundamentals

Electrophoresis fundamentals: essential theory and practice

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Bibliographic Details
Main Author:	Michov, Budin 1944- (Author)
Format:	Book
Language:	English
Published:	Berlin ; Boston De Gruyter [2022]
Series:	De Gruyter graduate
Subjects:	Elektrophorese Analytical Chemistry Biochemie Biochemistry Chemistry Electrophorese Electrophoresis Laboratory Medicine. Molekularbiologie Molekulare Medizin TB: Textbook
Online Access:	https://www.degruyter.com/isbn/9783110761627 Inhaltsverzeichnis
Physical Description:	XXXI, 490 Seiten Illustrationen, Diagramme
ISBN:	3110761629 9783110761627

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MARC


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adam_text	CONTENTS PREFACE - VII ABOUT THE AUTHOR - IX ABBREVIATIONS ---- XXIX 1 FUNDAMENTALS OF ELECTROPHORESIS - 1 OVERVIEW ON ELECTROPHORESIS - 1 SEPARATION MEDIA - 3 ELECTROPHORESIS RESOLUTION AND SHARPNESS - 3 DETECTION OF RESOLVED BANDS - 3 REFERENCES - 4 1.1 ELECTRIC DOUBLE LAYER OF A CHARGED PARTICLE - 5 1.1.1 ELECTRIC CHARGES OF POLYIONS - 5 1.1.2 MODEL OF HELMHOLTZ ---- 5 1.1.3 MODEL OF GOUY-CHAPMAN ------ 6 1.1.4 THEORY OF DEBYE-HIICKEL ------ 7 1.1.5 MODEL OF STERN ---- 7 1.1.6 TWO RADII AND TWO ELECTRIC POTENTIALS OF A CHARGED PARTICLE - 9 REFERENCES - 11 1.2 PROTEINS AND NUCLEIC ACIDS FORM POLYIONS IN SOLUTION - 13 1.2.1 STRUCTURE AND CONFORMATION OF PROTEINS ---- 13 1.2.1.1 MASSES AND ELECTRIC CHARGES OF PROTEINS - 13 1.2.1.2 ISOELECTRIC POINTS OF PROTEINS ---- 14 1.2.1.3 NATIVE AND DENATURED PROTEINS - 15 1.2.2 STRUCTURE AND CONFORMATION OF NUCLEIC ACIDS - 15 1.2.2.1 MASSES AND ELECTRIC CHARGES OF NUCLEIC ACIDS - 15 1.2.2.2 NATIVE AND DENATURED NUCLEIC ACIDS ----- 16 REFERENCES - 17 1.3 ELECTROPHORESIS IS RUNNING IN BUFFERS - 19 1.3.1 BUFFERS ---- 19 1.3.1.1 BUFFER CAPACITY ---- 20 1.3.2 BUFFERS USED IN ELECTROPHORESIS ----- 22 1.3.3 BIOLOGICAL BUFFERS ---- 23 REFERENCES -----26 XII CONTENTS 1.4 POLYIONS ARE MOVING IN ELECTRIC FIELD - 27 1.4.1 IONIC AND POLYIONIC MOBILITY ----- 27 1.4.1.1 EFFECTIVE MOBILITY ---- 28 1.4.2 EQUATIONS OF POLYIONIC MOBILITY ----- 29 1.4.2.1 EQUATION OF SMOLUCHOWSKI -----29 1.4.2.2 EQUATION OF HUCKEL ----- 29 1.4.2.3 HENRY S FUNCTION ----- 29 1.4.2.4 NEW EXPRESSION OF HENRY S FUNCTION ----- 30 1.4.2.5 EQUATION OF ONSAGER ---- 31 1.4.2. 6 EQUATION OF ROBINSON-STOKES ------ 32 1.4.2.7 PARAMETRIC EQUATION - 33 1.4.2.8 QUADRATIC EQUATION ----- 33 1.4.3 MOBILITIES OF IONS USED IN ELECTROPHORETIC METHODS ---- 33 1.4.3.1 CALCULATING THE MOBILITIES OF COMPOSED IONS ---- 35 REFERENCES ---- 37 1.5 ELECTROPHORESIS IS CARRIED OUT IN DIFFERENT SOLID MEDIA - 39 1.5.1 CELLULOSE ACETATE - 39 1.5.2 AGAROSE GEL - 39 1.5.3 POLYACRYLAMIDE GEL ---- 41 1.5.3.1 ACRYLAMIDE ----- 42 1.5.3.2 BISACRYLAMIDE ------42 1.5.3.3 THE MAGNITUDES TAND C ------ 42 1.5.3.4 ALTERNATIVE CROSS-LINKERS ------ 42 1.5.3.5 INITIATOR-CATALYST SYSTEMS ----- 43 1.5.3.6 COPOLYMERIZATION OF ACRYLAMIDE AND BIS ----- 45 1.5.3.7 HOMOGENEOUS POLYACRYLAMIDE GELS -------46 1.5.3.8 THIN AND ULTRATHIN POLYACRYLAMIDE GELS ---- 46 1.5.3.9 GRADIENT POLYACRYLAMIDE GELS ----- 47 REFERENCES - 47 1.6 GENERAL THEORY OF ELECTROPHORESIS - 49 1.6.1 WHAT IS THE POLYIONIC MOBILITY DEPENDING ON? ---- 50 1.6.1.1 INFLUENCE OF POLYIONIC NATURE - 50 1.6.1.2 INFLUENCE OF BUFFER ---- 50 1.6.1.3 INFLUENCE OF MEDIUM ---- 52 1.6.1.4 ELECTROOSMOSIS ---- 52 1.6.2 IONIC BOUNDARIES ---- 52 1.6.2.1 MOVING BOUNDARY ---- 52 1.6.2.2 STATIONARY BOUNDARY ---- 53 1.6.3 REGULATING FUNCTION ---- 53 1.6.4 DIFFUSION ---- 53 CONTENTS - - XIII 1.6.5 JOULE HEATING ---- 53 REFERENCES ---- 54 1.7 ELECTROPHORESIS INSTRUMENTATION - 55 1.7.1 ELECTROPHORESIS CELLS ---- 56 1.7.2 POWER SUPPLIES ---- 56 1.7.3 THERMOSTATS ---- 56 1.7.4 SCANNERS AND DENSITOMETERS ---- 56 1.7.5 GEL CASTING CASSETTES - 57 1.7.6 GRADIENT MAKERS - 57 1.7.7 BUFFER MIXERS ---- 58 1.7.8 BLOTTERS ---- 59 1.7.9 EQUIPMENT FOR SEMI-AUTOMATIC ELECTROPHORESIS - 59 REFERENCES - 60 1.8 CLASSIFICATION OF ELECTROPHORETIC METHODS - 61 1.8.1 ZONE ELECTROPHORESIS ---- 62 1.8.1.1 TISELIUS ELECTROPHORESIS ----- 62 1.8.1.2 CAPILLARY ELECTROPHORESIS ----- 62 1.8.1.3 FREE-FLOW ELECTROPHORESIS ---- 62 1.8.1.4 SOLID MEDIA ELECTROPHORESIS - 62 1.8.1.5 CELLULOSE ACETATE ELECTROPHORESIS - 63 1.8.1.6 AGAROSE GEL ELECTROPHORESIS - 63 1.8.1.7 PULSED-FIELD ELECTROPHORESIS - 63 1.8.1.8 IMMUNOELECTROPHORESIS AND IMMUNOFIXATION ----- 63 1.8.1.9 AFFINITY ELECTROPHORESIS ---- 64 1.8.1.10 POLYACRYLAMIDE GEL ELECTROPHORESIS ----- 64 1.8.1.11 IONTOPHORESIS ---- 64 1.8.2 ISOTACHOPHORESIS ----- 64 1.8.2.1 DISC-ELECTROPHORESIS ----- 64 1.8.2.2 NATIVE DISC-ELECTROPHORESIS ----- 65 1.8.2.3 SDS DISC-ELECTROPHORESIS ----- 65 1.8.3 ISOELECTRIC FOCUSING ---- 65 1.8.3.1 ISOELECTRIC FOCUSING WITH CARRIER AMPHOLYTES ---- 65 1.8.3.2 ISOELECTRIC FOCUSING IN IMMOBILIZED PH GRADIENTS ---- 65 1.8.3.3 TWO-DIMENSIONAL ELECTROPHORESIS ---- 66 1.8.4 DIELECTROPHORESIS ----- 66 1.8.4.1 DIELECTROPHORETIC FORCE - 66 1.8.4.2 DEP TECHNOLOGY ----- 67 1.8.4.3 APPLICATIONS OF DIELECTROPHORESIS ----- 68 REFERENCES - 68 XIV - CONTENTS 2 ELECTROPHORESIS OF PROTEINS - 71 REFERENCES - 71 2.1 CELLULOSE ACETATE ELECTROPHORESIS OF PROTEINS - 73 2.1.1 THEORY OF CELLULOSE ACETATE ELECTROPHORESIS OF PROTEINS - 73 2.1.2 PRACTICE OF CELLULOSE ACETATE ELECTROPHORESIS OF PROTEINS - 73 2.1.3 PROTOCOLS ---- 74 CELLULOSE ACETATE ELECTROPHORESIS OF SERUM PROTEINS - 74 REFERENCES - 75 2.2 AGAROSE GEL ELECTROPHORESIS OF PROTEINS - 77 2.2.1 THEORY OF AGAROSE GEL ELECTROPHORESIS OF PROTEINS ---- 78 2.2.2 AGAROSE GEL ELECTROPHORESIS OF SERUM PROTEINS ---- 78 2.2.2.1 ALBUMIN --- 80 INTERMEDIATE ZONE OF ALBUMIN-A R GLOBULINS - 80 2.2.2.2 ALPHA-1 GLOBULINS ----- 81 INTERMEDIATE ZONE OF A R A 2 -GLOBULINS - 81 2.2.2.3 ALPHA-2 GLOBULINS ----- 82 INTERMEDIATE ZONE OF A 2 -P-GLOBULINS - 82 2.2.2.4 BETA-GLOBULINS ----- 83 INTERMEDIATE ZONE OF P-Y-GLOBULINS - 84 2.2.2.5 GAMMA-GLOBULINS ----- 84 2.2.3 AGAROSE GEL ELECTROPHORESIS OF LIPOPROTEINS ----- 85 2.2.3.1 HIGH-DENSITY LIPOPROTEINS ----- 86 2.2.3.2 LOW-DENSITY LIPOPROTEINS ----- 86 2.2.3.3 INTERMEDIATE-DENSITY LIPOPROTEINS ----- 86 2.2.3.4 VERY-LOW-DENSITY LIPOPROTEINS ----- 86 2.2.3.5 CHYLOMICRONS ----- 86 2.2.3.6 HYPERLIPOPROTEINEMIAS ----- 87 2.2.4 AGAROSE GEL ELECTROPHORESIS OF HEMOGLOBINS ---- 89 2.2.4.1 NORMAL HEMOGLOBINS ------ 89 2.2.4.2 ABNORMAL AND PATHOLOGICAL HEMOGLOBINS ---- 90 SICKLE CELL DISEASE - 91 THALASSEMIAS - 91 2.2.4.3 RUNNING HEMOGLOBIN ELECTROPHORESIS - 93 2.2.5 AGAROSE GEL ELECTROPHORESIS OF CEREBROSPINAL FLUID PROTEINS - 94 2.2.6 ELECTROPHORESIS OF CREATINE KINASE ISOENZYMES ---- 95 2.2.7 ELECTROPHORESIS OF LACTATE DEHYDROGENASE ISOENZYMES - 96 2.2.8 AGAROSE GEL ELECTROPHORESIS OF URINARY PROTEINS - 97 2.2.9 PROTOCOLS ---- 98 AGAROSE GEL ELECTROPHORESIS OF SERUM PROTEINS - 98 AGAROSE GEL ELECTROPHORESIS OF LIPOPROTEINS - 100 CONTENTS - XV AGAROSE GEL ELECTROPHORESIS OF HEMOGLOBINS ---- 102 ALKALINE AGAROSE GEL ELECTROPHORESIS OF HEMOGLOBINS ---- 102 ACIDIC AGAROSE GEL ELECTROPHORESIS OF HEMOGLOBINS ---- 103 AGAROSE GEL ELECTROPHORESIS OF CEREBROSPINAL FLUID PROTEINS ---- 104 AGAROSE GEL ELECTROPHORESIS OF URINARY PROTEINS ---- 105 REFERENCES ---- 107 2.3 IMMUNOELECTROPHORESIS ---- 109 2.3.1 IMMUNODIFFUSION ELECTROPHORESIS ACCORDING TO GRABAR AND WILLIAMS - 110 2.3.2 ROCKET IMMUNOELECTROPHORESIS ACCORDING TO LAURELL ---- 110 2.3.3 IMMUNOFIXATION AND IMMUNOPRINTING ---- 111 2.3.4 PROTOCOLS ---- 112 IMMUNOFIXATION OF SERUM PROTEINS ---- 112 IMMUNOFIXATION OF BENCE JONES PROTEINS ---- 115 IMMUNOPROBING WITH AVIDIN-BIOTIN COUPLING TO SECONDARY ANTIBODY ---- 116 REFERENCES - 117 2.4 AFFINITY ELECTROPHORESIS---- 119 2.4.1 THEORY OF AFFINITY ELECTROPHORESIS ---- 119 2.4.2 LECTIN AFFINITY ELECTROPHORESIS ---- 119 2.4.2.1 ELECTROPHORESIS OF ALKALINE PHOSPHATASE ISOENZYMES ON LECTIN AGAROSE GELS ---- 120 2.4.3 SACCHARIDE AFFINITY ELECTROPHORESIS ---- 122 2.4.4 AFFINITY SUPPORTED MOLECULAR MATRIX ELECTROPHORESIS ---- 123 2.4.5 PHOSPHATE AFFINITY ELECTROPHORESIS - 123 2.4.6 CAPILLARY AFFINITY ELECTROPHORESIS ---- 125 2.4.7 AFFINITY-TRAP ELECTROPHORESIS ----- 125 2.4.8 CHARGE SHIFT ELECTROPHORESIS ----- 126 2.4.9 MOBILITY SHIFT ELECTROPHORESIS ----- 126 2.4.10 PROTOCOLS ---- 127 ELECTROPHORESIS OF ALP ISOENZYMES ON LECTIN AGAROSE GELS ---- 127 STAINING THE ALP ISOENZYMES ----- 128 REFERENCES ---- 129 2.5 POLYACRYLAMIDE GEL ZONE ELECTROPHORESIS OF PROTEINS - 131 2.5.1 HOMOGENEOUS GEL ZONE ELECTROPHORESIS OF PROTEINS - 131 2.5.1.1 THEORY OF POLYACRYLAMIDE GEL ZONE ELECTROPHORESIS OF PROTEINS ---- 132 2.5.1.2 MCLELLAN BUFFERS ----- 133 2.5.1.3 RUNNING ELECTROPHORESIS ---- 134 XVI - CONTENTS 2.5.2 2.5.2.1 2.5.2.2 2.5.23 2.53 2.5.4 GRADIENT GEL ZONE ELECTROPHORESIS OF PROTEINS - 134 THEORY OF GRADIENT GEL ZONE ELECTROPHORESIS ---- 135 FERGUSON PLOTS ---- 137 DETERMINATION OF STOKES RADII AND MASSES OF NATIVE PROTEINS - 138 BLUE NATIVE POLYACRYLAMIDE GEL ELECTROPHORESIS - 139 PROTOCOLS ---- 140 HORIZONTAL ELECTROPHORESIS OF PROTEINS ON GRADIENT GELS - 140 BLUE NATIVE POLYACRYLAMIDE GEL ELECTROPHORESIS - 142 PREPARATION OF DIALYZED CELL LYSATE - 142 CASTING GRADIENT BN GELS ---- 143 RUNNING BN ELECTROPHORESIS - 145 REFERENCES ---- 145 2.6 ISOTACHOPHORESIS OF PROTEINS ---- 147 2.6.1 2.6.1.1 THEORY OF ISOTACHOPHORESIS OF PROTEINS - 147 KOHLRAUSCH REGULATING FUNCTION ---- 147 REFERENCES ---- 150 2.7 DISC-ELECTROPHORESIS OF PROTEINS - 151 2.7.1 2.7.2 2.7.2.1 2.7.2.2 THEORY OF DISC-ELECTROPHORESIS ---- 152 NATIVE DISC-ELECTROPHORESIS ---- 153 DISC-ELECTROPHORESIS ACCORDING TO ORNSTEIN AND DAVIS - 153 BUFFER-GEL SYSTEMS FOR DISC-ELECTROPHORESIS ACCORDING TO ORNSTEIN AND DAVIS ---- 155 2.7.23 2.7.2.4 2.7.2.5 DISC-ELECTROPHORESIS ACCORDING TO ALLEN ETAL ---- 156 EFFECT OF HJERTEN ---- 156 BUFFER-GEL SYSTEMS FOR DISC-ELECTROPHORESIS ACCORDING TO ALLEN ETAL ---- 156 2.7.2.6 DISC-ELECTROPHORESIS IN ONE BUFFER AT TWO PH VALUES ACCORDING TO MICHOV ---- 157 2.7.2.7 2.7.2.8 THEORY OF DISC-ELECTROPHORESIS IN ONE BUFFER AT TWO PH VALUES - 157 BUFFER-GEL SYSTEMS FOR DISC-ELECTROPHORESIS IN ONE BUFFER AT TWO PH VALUES ---- 159 2.73 2.73.1 DENATURED SDS DISC-ELECTROPHORESIS ---- 161 DETERGENTS ---- 161 SODIUM DODECYL SULFATE - 163 SAMPLE PREPARATION FOR SDS POLYACRYLAMIDE GEL ELECTROPHORESIS ---- 165 NONREDUCING SAMPLE PREPARATION ---- 165 REDUCING SAMPLE PREPARATION ---- 165 REDUCING SAMPLE PREPARATION WITH ALKYLATION ---- 166 2.73.2 PRACTICE OF SDS DISC-ELECTROPHORESIS ---- 167 CONTENTS - XVII SDS DISC-ELECTROPHORESIS IN TRIS-CHLORIDE-GLYCINATE BUFFER SYSTEM ACCORDING TO LAEMMLI ---- 167 SDS DISC-ELECTROPHORESIS IN TRIS-ACETATE-TRICINEATE BUFFER SYSTEM ACCORDING TO SCHAGGER-JAGOW - 168 SDS DISC-ELECTROPHORESIS IN TRIS-FORMATE-TAURINATE BUFFER SYSTEM ACCORDING TO MICHOV - 169 SDS DISC-ELECTROPHORESIS IN ONE BUFFER AT TWO PH VALUES ACCORDING TO MICHOV ---- 170 SDS DISC-ELECTROPHORESIS IN GRADIENT GELS - 171 2.733 STAINING OF SDS PROTEIN BANDS - 171 DETERMINATION OF MOLECULAR MASSES OF SDS-DENATURED PROTEINS ----- 171 2.7.4 PROTOCOLS ---- 173 DISC-ELECTROPHORESIS IN ALKALINE BUFFER-GEL SYSTEM ACCORDING TO ORNSTEIN-DAVIS - 173 DISC-ELECTROPHORESIS IN ONE BUFFER AT TWO PH VALUES ACCORDING TO MICHOV ---- 176 SDS DISC-ELECTROPHORESIS ACCORDING TO LAEMMLI ---- 178 SDS ELECTROPHORESIS IN TRIS-FORMATE-TAURINATE BUFFER SYSTEM ACCORDING TO MICHOV ---- 180 COOMASSIE BRILLIANT BLUE R-250 STAINING OF PROTEINS RESOLVED IN SDS GELS ---- 183 REFERENCES - 184 2.8 ISOELECTRIC FOCUSING OF PROTEINS - 187 2.8.1 THEORY OF ISOELECTRIC FOCUSING ---- 188 2.8.2 ISOELECTRIC FOCUSING WITH CARRIER AMPHOLYTES ---- 189 2.8.2.1 PROPERTIES OF CARRIER AMPHOLYTES - 189 2.8.2.2 FORMATION OF A PH GRADIENT BY CARRIER AMPHOLYTES ---- 190 SEPARATOR ELECTROFOCUSING - 191 2.8.2.3 IEF WITH CARRIER AMPHOLYTES ON POLYACRYLAMIDE GELS ----- 191 THIN AND ULTRATHIN POLYACRYLAMIDE GELS FOR IEF WITH CARRIER AMPHOLYTES - 191 REHYDRATABLE POLYACRYLAMIDE GELS FOR IEF WITH CARRIER AMPHOLYTES ---- 191 2.8.2.4 SAMPLE PREPARATION AND APPLICATION ---- 192 2.8.2.5 ELECTRODE SOLUTIONS ----- 192 2.8.2.6 RUNNING ISOELECTRIC FOCUSING WITH CARRIER AMPHOLYTES ----- 193 2.8.3 ISOELECTRIC FOCUSING IN IMMOBILIZED PH GRADIENTS ---- 195 2.8.3.1 PROPERTIES OF IMMOBILINES ---- 196 2.8.3.2 CASTING IPG GELS ---- 197 REHYDRATABLE IPG GELS - 198 XVIII - - YY CONTENTS 2.8.3.3 2.8.4 2.8.5 RUNNING ISOELECTRIC FOCUSING ON IPG GELS - 198 ISOELECTRIC FOCUSING IN THE CLINICAL LABORATORY - 199 PROTOCOLS ---- 199 CASTING GELS FOR ISOELECTRIC FOCUSING WITH CARRIER AMPHOLYTES - 199 RUNNING ISOELECTRIC FOCUSING WITH CARRIER AMPHOLYTES ---- 200 CASTING IMMOBILINE GELS ---- 202 IEF WITH IPG GEL STRIPS ---- 202 REFERENCES - 206 2.9 FREE-FLOW ELECTROPHORESIS OF PROTEINS - 209 2.9.1 THEORY OF FREE-FLOW ELECTROPHORESIS ---- 209 2.9.2 TYPES OF FREE-FLOW ELECTROPHORESIS ---- 210 2.9.2.1 FREE-FLOW ZONE ELECTROPHORESIS ------ 211 2.9.2.2 FREE-FLOW ISOTACHOPHORESIS ------ 211 2.9.2.3 FREE-FLOW ISOELECTRIC FOCUSING ------ 212 2.9.3 DEVICE TECHNOLOGY OF FREE-FLOW ELECTROPHORESIS - 213 2.9.4 DETECTION SYSTEM OF FREE-FLOW ELECTROPHORESIS ----- 213 2.9.5 APPLICATIONS OF FREE-FLOW ELECTROPHORESIS ---- 214 2.9.6 PROTOCOLS ---- 214 FREE-FLOW ZONE ELECTROPHORESIS OF HUMAN T AND B LYMPHOCYTES ---- 214 REFERENCES ---- 215 2.10 CAPILLARY ELECTROPHORESIS OF PROTEINS - 217 2.10.1 THEORY OF CAPILLARY ELECTROPHORESIS OF PROTEINS - 217 2.10.2 INSTRUMENTATION ---- 218 2.10.2.1 COATING THE CAPILLARIES ---- 219 2.10.2.2 SIEVING MATRIX IN CAPILLARY ELECTROPHORESIS ---- 220 2.10.3 PRACTICE OF CAPILLARY ELECTROPHORESIS ----- 222 2.10.3.1 INJECTION ----- 222 2.10.3.2 SEPARATION ----- 222 2.10.3.3 DETECTION ----- 223 2.10.4 TYPES OF CAPILLARY ELECTROPHORESIS ----- 224 2.10.5 APPLICATIONS OF CAPILLARY ELECTROPHORESIS - 224 2.10.5.1 SERUM PROTEIN ANALYSIS - 224 2.10.5.2 HEMOGLOBINS ---- 225 2.10.5.3 ISOENZYMES ---- 226 2.10.5.4 IMMUNE COMPLEXES ----- 226 2.10.5.5 SINGLE CELL ANALYSIS ----- 227 2.10.6 PROTOCOLS ----- 227 CAPILLARY IEF OF PROTEINS ---- 227 REFERENCES ---- 228 CONTENTS - XIX 2.11 TWO-DIMENSIONAL ELECTROPHORESIS----- 231 2.11.1 THEORY OF 2D ELECTROPHORESIS ---- 231 2.11.2 ISOELECTRIC FOCUSING IN THE FIRST DIMENSION ---- 232 2.11.2.1 SAMPLE PREPARATION ---- 232 2.11.2.2 ISO-DALT AND IPG-DALT ---- 233 2.11.3 SDS DISC-ELECTROPHORESIS IN THE SECOND DIMENSION ---- 234 2.11.4 DETECTION AND EVALUATION OF PROTEINS IN 2D PHEROGRAMS ---- 236 2.11.4.1 AUTORADIOGRAPHY AND FLUOROGRAPHY ----- 236 2.11.4.2 TWO-DIMENSIONAL GEL IMAGE ANALYSIS ----- 236 2.11.5 PROTOCOLS ---- 237 TWO-DIMENSIONAL GEL ELECTROPHORESIS USING THE O FARRELL SYSTEM ---- 237 SAMPLE PREPARATION ---- 237 FIRST-DIMENSIONAL GELS (ISOELECTRIC FOCUSING) ---- 237 SECOND-DIMENSIONAL GELS (SDS ELECTROPHORESIS) ---- 238 REFERENCES - 239 2.12 PREPARATIVE ELECTROPHORESIS OF PROTEINS - 241 2.12.1 PREPARATIVE DISC-ELECTROPHORESIS ---- 241 2.12.1.1 ELUTION OF PROTEINS DURING ELECTROPHORESIS ---- 241 2.12.1.2 ELUTION OF PROTEINS AFTER ELECTROPHORESIS ---- 242 ELUTION BY DIFFUSION ---- 243 ELUTION BY GEL DISSOLVING ---- 243 ELECTROELUTION ---- 243 2.12.2 PREPARATIVE ISOELECTRIC FOCUSING ---- 245 2.12.2.1 PREPARATIVE IEF WITH CARRIER AMPHOLYTES IN GRANULATED GELS ---- 245 2.12.2.2 PREPARATIVE IEF IN IMMOBILIZED PH GRADIENTS ----- 247 2.12.2.3 RECYCLING ISOELECTRIC FOCUSING ---- 248 2.12.3 QPNC-PAGE ---- 248 2.12.4 PROTOCOLS ----- 249 QPNC-PAGE ---- 249 REFERENCES ---- 250 2.13 MICROCHIP ELECTROPHORESIS OF PROTEINS ---- 253 2.13.1 MICROCHIP MATERIALS 254 2.13.1.1 PDMS 254 2.13.1.2 PMMA ----- 255 2.13.1.3 PC ---- 255 2.13.2 MICROCHIP FABRICATION ------ 256 2.13.2.1 FABRICATION OF CHANNEL PLATE ---- 257 2.13.2.2 FABRICATION OF COVER PLATE ----- 258 2.13.2.3 WALL COATING ---- 258 XX - CONTENTS DYNAMIC WALL COATING ---- 258 PERMANENT WALL COATING ---- 259 2.13.2.4 BONDING THE PLATES ----- 259 2.13.3 ZONE ELECTROPHORESIS ON MICROCHIP ---- 261 2.13.3.1 FREE-FLOW ELECTROPHORESIS ON MICROCHIP ---- 261 2.13.3.2 AFFINITY AND IMMUNOELECTROPHORESIS ON MICROCHIP ---- 262 2.13.4 ISOTACHOPHORESIS ON MICROCHIP ----- 262 2.13.4.1 DISC-ELECTROPHORESIS ON MICROCHIP ----- 262 2.13.5 ISOELECTRIC FOCUSING ON MICROCHIP ----- 262 2.13.6 TWO-DIMENSIONAL ELECTROPHORESIS ON MICROCHIP - 263 2.13.7 PROTEIN SEPARATION TECHNIQUE ON MICROCHIP ---- 263 2.13.7.1 CONCENTRATING THE PROTEIN SAMPLES PRIOR TO MICROCHIP ELECTROPHORESIS ---- 263 2.13.7.2 RUNNING PROTEIN ELECTROPHORESIS ---- 264 2.13.7.3 DETECTING THE PROTEINS ---- 264 STAINING THE PROTEINS ---- 264 FLUORESCENCE DETECTION ---- 264 CHEMILUMINESCENCE DETECTION ---- 265 MASS SPECTROMETRY DETECTION ---- 265 2.13.8 MICROCHIPS IN CLINICAL DIAGNOSTICS ---- 265 REFERENCES ---- 266 2.14 BLOTTING OF PROTEINS ---- 271 2.14.1 THEORY OF PROTEIN BLOTTING ---- 271 2.14.2 BLOT MEMBRANES ---- 271 2.14.3 TRANSFER OF PROTEINS ---- 272 2.14.3.1 ELECTROTRANSFER OF PROTEINS ---- 272 TANK BLOTTING OF PROTEINS ---- 273 SEMIDRY BLOTTING OF PROTEINS ---- 273 2.14.3.2 CAPILLARY TRANSFER OF PROTEINS ---- 274 2.14.4 BLOCKING ---- 275 2.14.5 DETECTION ---- 276 2.14.5.1 DETECTION BY DYES ---- 276 2.14.5.2 DETECTION BY PROBES ---- 276 2.14.5.3 IMMUNOBLOTTING ---- 277 2.14.6 MAKING THE BLOT MEMBRANES TRANSPARENT ---- 277 2.14.7 BLOTTING TECHNIQUES ---- 278 2.14.7.1 WESTERN BLOTTING ---- 278 2.14.7.2 FAR-WESTERN BLOTTING ---- 279 2.14.7.3 SOUTHWESTERN BLOTTING ---- 279 2.14.7.4 NORTHWESTERN BLOTTING ---- 279 2.14.7.5 EASTERN BLOTTING ---- 280 CONTENTS - XXI 2.14.8 PROTOCOLS ---- 280 WESTERN BLOTTING ---- 280 SEMIDRY BLOTTING ---- 282 CAPILLARY BLOTTING ---- 283 INDIA INK STAINING OF PROTEINS ON MEMBRANE ---- 284 DETECTING PROTEINS BY IMMUNOBLOTTING ---- 285 IMMUNOPROBING WITH AVIDIN-BIOTIN COUPLING TO SECONDARY ANTIBODY ---- 286 REFERENCES - 287 2.15 EVALUATION OF PROTEIN PHEROGRAMS - 289 2.15.1 QUALITATIVE EVALUATION OF PROTEIN PHEROGRAMS - 289 2.15.1.1 FIXING OF PROTEINS ---- 290 2.15.1.2 STAINING OF PROTEINS ---- 290 ANIONIC DYE STAINING OF PROTEINS ---- 291 PONCEAU S STAINING OF PROTEINS ---- 291 AMIDO BLACK STAINING OF PROTEINS - 291 COOMASSIE BRILLIANT BLUE STAINING OF PROTEINS ---- 292 CONVENTIONAL COOMASSIE BRILLIANT BLUE STAINING OF PROTEINS ---- 292 COLLOIDAL COOMASSIE BRILLIANT BLUE STAINING OF PROTEINS ---- 293 INDIA INK STAINING OF PROTEINS ---- 293 COUNTER-IONIC DYE STAINING OF PROTEINS ---- 293 METAL STAINING OF PROTEINS - 294 SILVER STAINING OF PROTEINS - 294 CHELATE DYE STAINING OF PROTEINS ---- 295 STAINING OF GLYCOPROTEINS ---- 295 STAINING OF LIPOPROTEINS ---- 296 STAINING OF ENZYMES ---- 296 AUTORADIOGRAPHY OF PROTEINS ---- 297 FLUOROGRAPHY OF PROTEINS ---- 297 SYPRO RUBY STAINING OF PROTEINS ---- 298 DESTAINING OF GEL BACKGROUND ---- 298 DRYING OF GELS ---- 299 2.15.2 QUANTITATIVE EVALUATION OF A PROTEIN PHEROGRAM ---- 299 2.15.2.1 DENSITOMETRY ---- 299 DENSITOMETERS ---- 301 2.15.2.2 SCANNING ---- 302 2.15.3 PROTOCOLS ---- 302 COOMASSIE BRILLIANT BLUE STAINING OF PROTEINS IN POLYACRYLAMIDE GELS ---- 302 COLLOIDAL COOMASSIE STAINING ---- 303 RAPID SILVER STAINING ---- 304 XXII - CONTENTS SYPRO RUBY STAINING OF PROTEINS ---- 305 STAINING OF LIPOPROTEINS WITH SUDAN BLACK B ---- 305 REFERENCES ---- 306 2.16 PRECAST GELS FOR PROTEIN ELECTROPHORESIS. REHYDRATABLE GELS - 309 2.16.1 PRECAST AGAROSE GELS ---- 309 2.16.2 PRECAST POLYACRYLAMIDE GELS - 309 2.16.3 REHYDRATABLE POLYACRYLAMIDE GELS ---- 311 2.16.4 PROTOCOLS ---- 311 SILANIZATION OF GLASS PLATES - 311 CASTING AGAROSE GELS ON SUPPORT FILM BY CAPILLARY TECHNIQUE ---- 311 CASTING THIN PAA GELS BY CASSETTE TECHNIQUE ---- 312 CASTING ULTRATHIN PAA GELS BY FLAP TECHNIQUE ---- 314 REFERENCES - 315 3 ELECTROPHORESIS OF NUCLEIC ACIDS - 317 BUFFERS FOR ELECTROPHORESIS OF NUCLEIC ACIDS ---- 317 POLYMERASE CHAIN REACTION ---- 318 PROCEDURE ---- 318 APPLICATIONS ---- 320 SURFACE ELECTROPHORESIS ---- 321 REFERENCES ---- 323 3.1 AGAROSE GEL ELECTROPHORESIS OF NUCLEIC ACIDS - 325 3.1.1 ZONE POLYACRYLAMIDE GEL ELECTROPHORESIS OF NATIVE NUCLEIC ACIDS ---- 326 3.1.1.1 THEORY OF SIEVING MIGRATION ---- 326 FACTORS AFFECTING MIGRATION OF NUCLEIC ACIDS ---- 326 3.1.1.2 PRACTICE OF ZONE AGAROSE GEL ELECTROPHORESIS OF NATIVE NUCLEIC ACIDS ---- 327 SUBMARINE ELECTROPHORESIS OF NUCLEIC ACIDS ---- 327 RESTRICTION FRAGMENT LENGTH POLYMORPHISM ---- 328 VARIABLE NUMBER TANDEM REPEAT ---- 329 DNA PROFILING ---- 329 PATERNITY OR MATERNITY TESTING FOR CHILD ---- 330 INTERPRETATION OF DNA TEST RESULTS ---- 331 3.1.2 ZONE AGAROSE GEL ELECTROPHORESIS OF DENATURED NUCLEIC ACIDS - 332 3.1.3 DNA SEQUENCING ---- 332 3.1.3.1 MAXAM-GILBERT SEQUENCING METHOD ---- 332 3.1.3.2 SANGER SEQUENCING METHOD ----- 333 3.1.3.3 SINGLE-STRAND CONFORMATION POLYMORPHISM METHOD ---- 334 3.1.3.4 DNASE FOOTPRINTING ASSAY ----- 336 CONTENTS - - XXIII 3.1.3.5 NUCLEASE PROTECTION ASSAY ---- 337 SJ-NUDEASE PROTECTION ASSAY ---- 337 3.1.4 RNA SEPARATION ---- 338 3.1.4.1 PRIMER EXTENSION ASSAY ----- 339 3.1.5 PROTOCOLS ---- 339 NATIVE DNA ELECTROPHORESIS ON AGAROSE GELS ---- 339 SANGER SEQUENCING REACTIONS USING TAQ DNA POLYMERASE ---- 340 REFERENCES - 341 3.2 PULSED-FIELD GEL ELECTROPHORESIS OF NUCLEIC ACIDS - 343 3.2.1 THEORY OF PULSED-FIELD GEL ELECTROPHORESIS OF NUCLEIC ACIDS ---- 343 3.2.2 TYPES OF PULSED-FIELD GEL ELECTROPHORESIS ---- 344 3.2.3 MAPPING THE HUMAN GENOME ---- 345 3.2.3.1 STR ANALYSIS ---- 345 3.2.3.2 AMPFLP ---- 345 3.2.3.3 DNA FAMILY RELATIONSHIP ANALYSIS ----- 346 3.2.3.4 Y-CHROMOSOME ANALYSIS ----- 346 3.2.3.5 MITOCHONDRIAL ANALYSIS ----- 346 3.2.4 PROTOCOLS ----- 346 PULSED-FIELD GEL ELECTROPHORESIS OF CHROMOSOMAL DNA---- 346 REFERENCES ---- 347 3.3 CAPILLARY ELECTROPHORESIS OF NUCLEIC ACIDS - 349 3.3.1 THEORY OF CAPILLARY ELECTROPHORESIS OF NUCLEIC ACIDS ---- 349 3.3.2 INSTRUMENTATION FOR CAPILLARY ELECTROPHORESIS ---- 350 3.3. 2.1 CAPILLARIES ----- 350 3.3.2.2 COATING POLYMERS ----- 350 3.3.2.3 GELS ----- 351 3.3.3 RUNNING CAPILLARY ELECTROPHORESIS - 352 3.3.3.1 INJECTION ---- 352 3.3.3.2 SEPARATION ----- 352 3.3.3.3 DETECTION ----- 352 3.3.4 PULSED-FIELD CAPILLARY ELECTROPHORESIS ---- 353 3.3.5 APPLICATIONS OF CAPILLARY ELECTROPHORESIS ---- 353 3.3.6 PROTOCOLS ---- 354 QUANTITATIVE PCR ANALYSIS ---- 354 REFERENCES - 354 3.4 POLYACRYLAMIDE GEL ELECTROPHORESIS OF NUCLEIC ACIDS - 357 3.4.1 ZONE POLYACRYLAMIDE GEL ELECTROPHORESIS OF NATIVE NUCLEIC ACIDS ----- 357 XXIV - CONTENTS 3.4.1.1 PRACTICE OF ZONE POLYACRYLAMIDE GEL ELECTROPHORESIS OF NATIVE NUCLEIC ACIDS ---- 358 3.4.2 DISC-ELECTROPHORESIS OF NATIVE NUCLEIC ACIDS ----- 360 3.4.2.1 THEORY OF DISC-ELECTROPHORESIS OF NATIVE NUCLEIC ACIDS ----- 360 3.4.2.2 RUNNING DISC-ELECTROPHORESIS OF NATIVE NUCLEIC ACIDS - 360 3.4.2.3 DISC-ELECTROPHORESIS OF DOUBLE-STRANDED PCR PRODUCTS ----- 361 3.4.3 ELECTROPHORETIC MOBILITY SHIFT ASSAY ---- 363 3.4.4 CLINICAL APPLICATIONS ---- 364 3.4.5 PROTOCOLS ---- 364 DNA DISC-ELECTROPHORESIS IN TRIS-TAURINATE BUFFER AT TWO PH VALUES ACCORDING TO MICHOV ---- 364 SEPARATION OF PCR PRODUCTS BY POLYACRYLAMIDE GEL DISC ELECTROPHORESIS ACCORDING TO MICHOV ---- 366 ELECTROPHORETIC MOBILITY SHIFT ASSAY ---- 367 ELECTROELUTION OF SMALL DNA FRAGMENTS FROM POLYACRYLAMIDE GEL ---- 368 REFERENCES ---- 368 3.5 MICROCHIP ELECTROPHORESIS OF NUCLEIC ACIDS - 371 3.5.1 THEORY OF MICROCHIP ELECTROPHORESIS OF NUCLEIC ACIDS ---- 371 3.5.2 CONSTRUCTION OF A MICROCHIP FOR ELECTROPHORESIS OF NUCLEIC ACIDS ---- 371 3.5.2.1 POLYMERS ----- 372 3.5.3 RUNNING MICROCHIP ELECTROPHORESIS OF NUCLEIC ACIDS ---- 373 3.5.4 APPLICATIONS OF DNA MICROCHIP ELECTROPHORESIS ---- 373 REFERENCES ---- 373 3.6 BLOTTING OF NUCLEIC ACIDS ---- 375 3.6.1 BLOTTING PRINCIPLES ---- 375 3.6.1.1 TRANSFER ---- 375 DIFFUSION TRANSFER --- 376 CAPILLARY TRANSFER ---- 376 VACUUM TRANSFER ---- 376 ELECTROTRANSFER ---- 377 3.6.1.2 BLOCKING ----- 378 3.6.1.3 DETECTION BY PROBES, DYES, AND AUTORADIOGRAPHY ----- 378 3.6.2 SOUTHERN BLOTTING ------- 379 3.6.3 NORTHERN BLOTTING ------- 380 3.6.4 MIDDLE-EASTERN BLOTTING ---- 381 3.6.5 PROTOCOLS ---- 382 SOUTHERN BLOTTING BY DOWNWARD CAPILLARY TRANSFER ---- 382 ELECTROBLOTTING OF POLYACRYLAMIDE GEL ONTO NYLON MEMBRANE ---- 383 REFERENCES ---- 384 CONTENTS - - XXV 3.7 EVALUATION OF NUCLEIC ACID PHEROGRAMS ---- 385 3.7.1 COUNTER-ION DYE STAINING OF NUCLEIC ACIDS ---- 385 3.7.2 SILVER STAINING OF NUCLEIC ACIDS ---- 385 3.7.3 FLUORESCENCE METHODS FOR DETECTING NUCLEIC ACIDS 386 3.7.4 AUTORADIOGRAPHY OF NUCLEIC ACIDS --- 389 3.7.5 LABELING OF NUCLEIC ACIDS WITH PROTEINS ---- 390 3.7.6 ABSORPTION SPECTROSCOPY OF NUCLEIC ACIDS ----- 391 3.7.7 PROTOCOLS ---- 391 DETECTION OF DNA AND RNA IN GEL USING ETHIDIUM BROMIDE ---- 391 FAST AND SENSITIVE SILVER STAINING OF DNA BANDS ACCORDING TO HAN ETAL. [34] ---- 392 AUTORADIOGRAPHY OF RADIOLABELED DNA IN GELS AND BLOTS ---- 392 REFERENCES - 394 3.8 PRECAST GELS FOR NUCLEIC ACID ELECTROPHORESIS - 395 3.8.1 PRECAST AGAROSE GELS ---- 395 3.8.2 PRECAST POLYACRYLAMIDE GELS ---- 395 3.8.3 PROTOCOLS ---- 396 CASTING MINI AND MIDI-AGAROSE GELS ---- 396 4 IONTOPHORESIS --- 397 4.1 THEORY OF IONTOPHORESIS ---- 397 4.2 FACTORS AFFECTING IONTOPHORESIS ---- 398 4.2.1 PHYSICOCHEMICAL FACTORS ---- 398 4.2.2 BIOLOGICAL FACTORS ---- 399 4.3 CALCULATING THE IONTOPHORETIC CURRENT ---- 399 4.4 IONTOPHORESIS DEVICE ---- 399 4.5 DIAGNOSTIC IONTOPHORESIS ---- 400 4.6 THERAPEUTIC IONTOPHORESIS ---- 400 4.6.1 TRANSDERMAL IONTOPHORESIS ---- 400 HYPERHIDROSIS IONTOPHORESIS ---- 401 OTHER TRANSDERMAL APPLICATIONS - 401 4.6.2 OCULAR IONTOPHORESIS ---- 402 REFERENCES ---- 403 5 HISTORY OF ELECTROPHORESIS AND IONTOPHORESIS---- 405 5.1 HISTORY OF ELECTROPHORESIS ---- 405 5.1.1 DISCOVERY OF ELECTROPHORESIS ---- 405 5.1.2 ZONE ELECTROPHORESIS OF TISELIUS ---- 406 5.1.3 PAPER AND CELLULOSE ACETATE ELECTROPHORESIS ---- 406 5.1.4 GEL ELECTROPHORESIS ---- 406 AGAROSE GEL ELECTROPHORESIS - 406 XXVI - CONTENTS POLYACRYALAMIDE GEL ELECTROPHORESIS - 407 5.1.5 5.1.6 5.1.7 ISOELECTRIC FOCUSING - 407 TWO-DIMENSIONAL ELECTROPHORESIS ---- 407 BLOTTING ---- 408 BLOTTING OF PROTEINS - 408 BLOTTING OF NUCLEIC ACIDS ---- 409 5.1.8 5.1.9 5.2 5.2.1 STAINING METHODS ---- 409 OUTLINE HISTORY OF ELECTROPHORESIS - 409 HISTORY OF IONTOPHORESIS ---- 413 OUTLINE HISTORY OF IONTOPHORESIS - 414 REFERENCES ---- 415 6 6.1 6.2 6.3 6.4 6.5 TROUBLESHOOTING ---- 421 PROTEIN ELECTROPHORESIS TROUBLESHOOTING ---- 421 IEF TROUBLESHOOTING ---- 421 NUCLEIC ACID ELECTROPHORESIS TROUBLESHOOTING - 427 BLOTTING TROUBLESHOOTING - 431 IONTOPHORESIS TROUBLESHOOTING - 433 PROBLEMS ---- 435 1 FUNDAMENTALS OF ELECTROPHORESIS ----- 435 2 ELECTROPHORESIS OF PROTEINS ---- 436 3 ELECTROPHORESIS OF NUCLEIC ACIDS ----- 438 SOLUTION OF PROBLEMS - 441 1 FUNDAMENTALS OF ELECTROPHORESIS - 441 2 ELECTROPHORESIS OF PROTEINS ---- 442 3 ELECTROPHORESIS OF NUCLEIC ACIDS ---- 445 REAGENTS FOR ELECTROPHORESIS - 447 RECIPES FOR ELECTROPHORESIS SOLUTIONS - 459 BUFFERS ---- 459 SOLUTIONS FOR AGAROSE GEL ELECTROPHORESIS ---- 463 SOLUTIONS FOR AFFINITY ELECTROPHORESIS ---- 465 SOLUTIONS FOR NATIVE DISC-ELECTROPHORESIS ---- 466 SOLUTIONS FOR SDS DISC-ELECTROPHORESIS ---- 467 SOLUTIONS FOR IEF ---- 468 BLOTTING SOLUTIONS ---- 469 FIXATIVE SOLUTIONS ---- 470 STAINING SOLUTIONS ---- 471 DESTAINING SOLUTIONS ---- 472 CONTENTS - - XXVII SILVER STAINING SOLUTIONS ---- 472 OTHER SOLUTIONS ---- 473 SI UNITS AND PHYSICAL CONSTANTS USED IN ELECTROPHORESIS - 475 REFERENCES ---- 479 ELECTROPHORESIS TERMS - 481 INDEX ---- 485
adam_txt	CONTENTS PREFACE - VII ABOUT THE AUTHOR - IX ABBREVIATIONS ---- XXIX 1 FUNDAMENTALS OF ELECTROPHORESIS - 1 OVERVIEW ON ELECTROPHORESIS - 1 SEPARATION MEDIA - 3 ELECTROPHORESIS RESOLUTION AND SHARPNESS - 3 DETECTION OF RESOLVED BANDS - 3 REFERENCES - 4 1.1 ELECTRIC DOUBLE LAYER OF A CHARGED PARTICLE - 5 1.1.1 ELECTRIC CHARGES OF POLYIONS - 5 1.1.2 MODEL OF HELMHOLTZ ---- 5 1.1.3 MODEL OF GOUY-CHAPMAN ------ 6 1.1.4 THEORY OF DEBYE-HIICKEL ------ 7 1.1.5 MODEL OF STERN ---- 7 1.1.6 TWO RADII AND TWO ELECTRIC POTENTIALS OF A CHARGED PARTICLE - 9 REFERENCES - 11 1.2 PROTEINS AND NUCLEIC ACIDS FORM POLYIONS IN SOLUTION - 13 1.2.1 STRUCTURE AND CONFORMATION OF PROTEINS ---- 13 1.2.1.1 MASSES AND ELECTRIC CHARGES OF PROTEINS - 13 1.2.1.2 ISOELECTRIC POINTS OF PROTEINS ---- 14 1.2.1.3 NATIVE AND DENATURED PROTEINS - 15 1.2.2 STRUCTURE AND CONFORMATION OF NUCLEIC ACIDS - 15 1.2.2.1 MASSES AND ELECTRIC CHARGES OF NUCLEIC ACIDS - 15 1.2.2.2 NATIVE AND DENATURED NUCLEIC ACIDS ----- 16 REFERENCES - 17 1.3 ELECTROPHORESIS IS RUNNING IN BUFFERS - 19 1.3.1 BUFFERS ---- 19 1.3.1.1 BUFFER CAPACITY ---- 20 1.3.2 BUFFERS USED IN ELECTROPHORESIS ----- 22 1.3.3 BIOLOGICAL BUFFERS ---- 23 REFERENCES -----26 XII CONTENTS 1.4 POLYIONS ARE MOVING IN ELECTRIC FIELD - 27 1.4.1 IONIC AND POLYIONIC MOBILITY ----- 27 1.4.1.1 EFFECTIVE MOBILITY ---- 28 1.4.2 EQUATIONS OF POLYIONIC MOBILITY ----- 29 1.4.2.1 EQUATION OF SMOLUCHOWSKI -----29 1.4.2.2 EQUATION OF HUCKEL ----- 29 1.4.2.3 HENRY ' S FUNCTION ----- 29 1.4.2.4 NEW EXPRESSION OF HENRY ' S FUNCTION ----- 30 1.4.2.5 EQUATION OF ONSAGER ---- 31 1.4.2. 6 EQUATION OF ROBINSON-STOKES ------ 32 1.4.2.7 PARAMETRIC EQUATION - 33 1.4.2.8 QUADRATIC EQUATION ----- 33 1.4.3 MOBILITIES OF IONS USED IN ELECTROPHORETIC METHODS ---- 33 1.4.3.1 CALCULATING THE MOBILITIES OF COMPOSED IONS ---- 35 REFERENCES ---- 37 1.5 ELECTROPHORESIS IS CARRIED OUT IN DIFFERENT SOLID MEDIA - 39 1.5.1 CELLULOSE ACETATE - 39 1.5.2 AGAROSE GEL - 39 1.5.3 POLYACRYLAMIDE GEL ---- 41 1.5.3.1 ACRYLAMIDE ----- 42 1.5.3.2 BISACRYLAMIDE ------42 1.5.3.3 THE MAGNITUDES TAND C ------ 42 1.5.3.4 ALTERNATIVE CROSS-LINKERS ------ 42 1.5.3.5 INITIATOR-CATALYST SYSTEMS ----- 43 1.5.3.6 COPOLYMERIZATION OF ACRYLAMIDE AND BIS ----- 45 1.5.3.7 HOMOGENEOUS POLYACRYLAMIDE GELS -------46 1.5.3.8 THIN AND ULTRATHIN POLYACRYLAMIDE GELS ---- 46 1.5.3.9 GRADIENT POLYACRYLAMIDE GELS ----- 47 REFERENCES - 47 1.6 GENERAL THEORY OF ELECTROPHORESIS - 49 1.6.1 WHAT IS THE POLYIONIC MOBILITY DEPENDING ON? ---- 50 1.6.1.1 INFLUENCE OF POLYIONIC NATURE - 50 1.6.1.2 INFLUENCE OF BUFFER ---- 50 1.6.1.3 INFLUENCE OF MEDIUM ---- 52 1.6.1.4 ELECTROOSMOSIS ---- 52 1.6.2 IONIC BOUNDARIES ---- 52 1.6.2.1 MOVING BOUNDARY ---- 52 1.6.2.2 STATIONARY BOUNDARY ---- 53 1.6.3 REGULATING FUNCTION ---- 53 1.6.4 DIFFUSION ---- 53 CONTENTS - - XIII 1.6.5 JOULE HEATING ---- 53 REFERENCES ---- 54 1.7 ELECTROPHORESIS INSTRUMENTATION - 55 1.7.1 ELECTROPHORESIS CELLS ---- 56 1.7.2 POWER SUPPLIES ---- 56 1.7.3 THERMOSTATS ---- 56 1.7.4 SCANNERS AND DENSITOMETERS ---- 56 1.7.5 GEL CASTING CASSETTES - 57 1.7.6 GRADIENT MAKERS - 57 1.7.7 BUFFER MIXERS ---- 58 1.7.8 BLOTTERS ---- 59 1.7.9 EQUIPMENT FOR SEMI-AUTOMATIC ELECTROPHORESIS - 59 REFERENCES - 60 1.8 CLASSIFICATION OF ELECTROPHORETIC METHODS - 61 1.8.1 ZONE ELECTROPHORESIS ---- 62 1.8.1.1 TISELIUS ELECTROPHORESIS ----- 62 1.8.1.2 CAPILLARY ELECTROPHORESIS ----- 62 1.8.1.3 FREE-FLOW ELECTROPHORESIS ---- 62 1.8.1.4 SOLID MEDIA ELECTROPHORESIS - 62 1.8.1.5 CELLULOSE ACETATE ELECTROPHORESIS - 63 1.8.1.6 AGAROSE GEL ELECTROPHORESIS - 63 1.8.1.7 PULSED-FIELD ELECTROPHORESIS - 63 1.8.1.8 IMMUNOELECTROPHORESIS AND IMMUNOFIXATION ----- 63 1.8.1.9 AFFINITY ELECTROPHORESIS ---- 64 1.8.1.10 POLYACRYLAMIDE GEL ELECTROPHORESIS ----- 64 1.8.1.11 IONTOPHORESIS ---- 64 1.8.2 ISOTACHOPHORESIS ----- 64 1.8.2.1 DISC-ELECTROPHORESIS ----- 64 1.8.2.2 NATIVE DISC-ELECTROPHORESIS ----- 65 1.8.2.3 SDS DISC-ELECTROPHORESIS ----- 65 1.8.3 ISOELECTRIC FOCUSING ---- 65 1.8.3.1 ISOELECTRIC FOCUSING WITH CARRIER AMPHOLYTES ---- 65 1.8.3.2 ISOELECTRIC FOCUSING IN IMMOBILIZED PH GRADIENTS ---- 65 1.8.3.3 TWO-DIMENSIONAL ELECTROPHORESIS ---- 66 1.8.4 DIELECTROPHORESIS ----- 66 1.8.4.1 DIELECTROPHORETIC FORCE - 66 1.8.4.2 DEP TECHNOLOGY ----- 67 1.8.4.3 APPLICATIONS OF DIELECTROPHORESIS ----- 68 REFERENCES - 68 XIV - CONTENTS 2 ELECTROPHORESIS OF PROTEINS - 71 REFERENCES - 71 2.1 CELLULOSE ACETATE ELECTROPHORESIS OF PROTEINS - 73 2.1.1 THEORY OF CELLULOSE ACETATE ELECTROPHORESIS OF PROTEINS - 73 2.1.2 PRACTICE OF CELLULOSE ACETATE ELECTROPHORESIS OF PROTEINS - 73 2.1.3 PROTOCOLS ---- 74 CELLULOSE ACETATE ELECTROPHORESIS OF SERUM PROTEINS - 74 REFERENCES - 75 2.2 AGAROSE GEL ELECTROPHORESIS OF PROTEINS - 77 2.2.1 THEORY OF AGAROSE GEL ELECTROPHORESIS OF PROTEINS ---- 78 2.2.2 AGAROSE GEL ELECTROPHORESIS OF SERUM PROTEINS ---- 78 2.2.2.1 ALBUMIN --- 80 INTERMEDIATE ZONE OF ALBUMIN-A R GLOBULINS - 80 2.2.2.2 ALPHA-1 GLOBULINS ----- 81 INTERMEDIATE ZONE OF A R A 2 -GLOBULINS - 81 2.2.2.3 ALPHA-2 GLOBULINS ----- 82 INTERMEDIATE ZONE OF A 2 -P-GLOBULINS - 82 2.2.2.4 BETA-GLOBULINS ----- 83 INTERMEDIATE ZONE OF P-Y-GLOBULINS - 84 2.2.2.5 GAMMA-GLOBULINS ----- 84 2.2.3 AGAROSE GEL ELECTROPHORESIS OF LIPOPROTEINS ----- 85 2.2.3.1 HIGH-DENSITY LIPOPROTEINS ----- 86 2.2.3.2 LOW-DENSITY LIPOPROTEINS ----- 86 2.2.3.3 INTERMEDIATE-DENSITY LIPOPROTEINS ----- 86 2.2.3.4 VERY-LOW-DENSITY LIPOPROTEINS ----- 86 2.2.3.5 CHYLOMICRONS ----- 86 2.2.3.6 HYPERLIPOPROTEINEMIAS ----- 87 2.2.4 AGAROSE GEL ELECTROPHORESIS OF HEMOGLOBINS ---- 89 2.2.4.1 NORMAL HEMOGLOBINS ------ 89 2.2.4.2 ABNORMAL AND PATHOLOGICAL HEMOGLOBINS ---- 90 SICKLE CELL DISEASE - 91 THALASSEMIAS - 91 2.2.4.3 RUNNING HEMOGLOBIN ELECTROPHORESIS - 93 2.2.5 AGAROSE GEL ELECTROPHORESIS OF CEREBROSPINAL FLUID PROTEINS - 94 2.2.6 ELECTROPHORESIS OF CREATINE KINASE ISOENZYMES ---- 95 2.2.7 ELECTROPHORESIS OF LACTATE DEHYDROGENASE ISOENZYMES - 96 2.2.8 AGAROSE GEL ELECTROPHORESIS OF URINARY PROTEINS - 97 2.2.9 PROTOCOLS ---- 98 AGAROSE GEL ELECTROPHORESIS OF SERUM PROTEINS - 98 AGAROSE GEL ELECTROPHORESIS OF LIPOPROTEINS - 100 CONTENTS - XV AGAROSE GEL ELECTROPHORESIS OF HEMOGLOBINS ---- 102 ALKALINE AGAROSE GEL ELECTROPHORESIS OF HEMOGLOBINS ---- 102 ACIDIC AGAROSE GEL ELECTROPHORESIS OF HEMOGLOBINS ---- 103 AGAROSE GEL ELECTROPHORESIS OF CEREBROSPINAL FLUID PROTEINS ---- 104 AGAROSE GEL ELECTROPHORESIS OF URINARY PROTEINS ---- 105 REFERENCES ---- 107 2.3 IMMUNOELECTROPHORESIS ---- 109 2.3.1 IMMUNODIFFUSION ELECTROPHORESIS ACCORDING TO GRABAR AND WILLIAMS - 110 2.3.2 ROCKET IMMUNOELECTROPHORESIS ACCORDING TO LAURELL ---- 110 2.3.3 IMMUNOFIXATION AND IMMUNOPRINTING ---- 111 2.3.4 PROTOCOLS ---- 112 IMMUNOFIXATION OF SERUM PROTEINS ---- 112 IMMUNOFIXATION OF BENCE JONES PROTEINS ---- 115 IMMUNOPROBING WITH AVIDIN-BIOTIN COUPLING TO SECONDARY ANTIBODY ---- 116 REFERENCES - 117 2.4 AFFINITY ELECTROPHORESIS---- 119 2.4.1 THEORY OF AFFINITY ELECTROPHORESIS ---- 119 2.4.2 LECTIN AFFINITY ELECTROPHORESIS ---- 119 2.4.2.1 ELECTROPHORESIS OF ALKALINE PHOSPHATASE ISOENZYMES ON LECTIN AGAROSE GELS ---- 120 2.4.3 SACCHARIDE AFFINITY ELECTROPHORESIS ---- 122 2.4.4 AFFINITY SUPPORTED MOLECULAR MATRIX ELECTROPHORESIS ---- 123 2.4.5 PHOSPHATE AFFINITY ELECTROPHORESIS - 123 2.4.6 CAPILLARY AFFINITY ELECTROPHORESIS ---- 125 2.4.7 AFFINITY-TRAP ELECTROPHORESIS ----- 125 2.4.8 CHARGE SHIFT ELECTROPHORESIS ----- 126 2.4.9 MOBILITY SHIFT ELECTROPHORESIS ----- 126 2.4.10 PROTOCOLS ---- 127 ELECTROPHORESIS OF ALP ISOENZYMES ON LECTIN AGAROSE GELS ---- 127 STAINING THE ALP ISOENZYMES ----- 128 REFERENCES ---- 129 2.5 POLYACRYLAMIDE GEL ZONE ELECTROPHORESIS OF PROTEINS - 131 2.5.1 HOMOGENEOUS GEL ZONE ELECTROPHORESIS OF PROTEINS - 131 2.5.1.1 THEORY OF POLYACRYLAMIDE GEL ZONE ELECTROPHORESIS OF PROTEINS ---- 132 2.5.1.2 MCLELLAN BUFFERS ----- 133 2.5.1.3 RUNNING ELECTROPHORESIS ---- 134 XVI - CONTENTS 2.5.2 2.5.2.1 2.5.2.2 2.5.23 2.53 2.5.4 GRADIENT GEL ZONE ELECTROPHORESIS OF PROTEINS - 134 THEORY OF GRADIENT GEL ZONE ELECTROPHORESIS ---- 135 FERGUSON PLOTS ---- 137 DETERMINATION OF STOKES RADII AND MASSES OF NATIVE PROTEINS - 138 BLUE NATIVE POLYACRYLAMIDE GEL ELECTROPHORESIS - 139 PROTOCOLS ---- 140 HORIZONTAL ELECTROPHORESIS OF PROTEINS ON GRADIENT GELS - 140 BLUE NATIVE POLYACRYLAMIDE GEL ELECTROPHORESIS - 142 PREPARATION OF DIALYZED CELL LYSATE - 142 CASTING GRADIENT BN GELS ---- 143 RUNNING BN ELECTROPHORESIS - 145 REFERENCES ---- 145 2.6 ISOTACHOPHORESIS OF PROTEINS ---- 147 2.6.1 2.6.1.1 THEORY OF ISOTACHOPHORESIS OF PROTEINS - 147 KOHLRAUSCH REGULATING FUNCTION ---- 147 REFERENCES ---- 150 2.7 DISC-ELECTROPHORESIS OF PROTEINS - 151 2.7.1 2.7.2 2.7.2.1 2.7.2.2 THEORY OF DISC-ELECTROPHORESIS ---- 152 NATIVE DISC-ELECTROPHORESIS ---- 153 DISC-ELECTROPHORESIS ACCORDING TO ORNSTEIN AND DAVIS - 153 BUFFER-GEL SYSTEMS FOR DISC-ELECTROPHORESIS ACCORDING TO ORNSTEIN AND DAVIS ---- 155 2.7.23 2.7.2.4 2.7.2.5 DISC-ELECTROPHORESIS ACCORDING TO ALLEN ETAL ---- 156 EFFECT OF HJERTEN ---- 156 BUFFER-GEL SYSTEMS FOR DISC-ELECTROPHORESIS ACCORDING TO ALLEN ETAL ---- 156 2.7.2.6 DISC-ELECTROPHORESIS IN ONE BUFFER AT TWO PH VALUES ACCORDING TO MICHOV ---- 157 2.7.2.7 2.7.2.8 THEORY OF DISC-ELECTROPHORESIS IN ONE BUFFER AT TWO PH VALUES - 157 BUFFER-GEL SYSTEMS FOR DISC-ELECTROPHORESIS IN ONE BUFFER AT TWO PH VALUES ---- 159 2.73 2.73.1 DENATURED SDS DISC-ELECTROPHORESIS ---- 161 DETERGENTS ---- 161 SODIUM DODECYL SULFATE - 163 SAMPLE PREPARATION FOR SDS POLYACRYLAMIDE GEL ELECTROPHORESIS ---- 165 NONREDUCING SAMPLE PREPARATION ---- 165 REDUCING SAMPLE PREPARATION ---- 165 REDUCING SAMPLE PREPARATION WITH ALKYLATION ---- 166 2.73.2 PRACTICE OF SDS DISC-ELECTROPHORESIS ---- 167 CONTENTS - XVII SDS DISC-ELECTROPHORESIS IN TRIS-CHLORIDE-GLYCINATE BUFFER SYSTEM ACCORDING TO LAEMMLI ---- 167 SDS DISC-ELECTROPHORESIS IN TRIS-ACETATE-TRICINEATE BUFFER SYSTEM ACCORDING TO SCHAGGER-JAGOW - 168 SDS DISC-ELECTROPHORESIS IN TRIS-FORMATE-TAURINATE BUFFER SYSTEM ACCORDING TO MICHOV - 169 SDS DISC-ELECTROPHORESIS IN ONE BUFFER AT TWO PH VALUES ACCORDING TO MICHOV ---- 170 SDS DISC-ELECTROPHORESIS IN GRADIENT GELS - 171 2.733 STAINING OF SDS PROTEIN BANDS - 171 DETERMINATION OF MOLECULAR MASSES OF SDS-DENATURED PROTEINS ----- 171 2.7.4 PROTOCOLS ---- 173 DISC-ELECTROPHORESIS IN ALKALINE BUFFER-GEL SYSTEM ACCORDING TO ORNSTEIN-DAVIS - 173 DISC-ELECTROPHORESIS IN ONE BUFFER AT TWO PH VALUES ACCORDING TO MICHOV ---- 176 SDS DISC-ELECTROPHORESIS ACCORDING TO LAEMMLI ---- 178 SDS ELECTROPHORESIS IN TRIS-FORMATE-TAURINATE BUFFER SYSTEM ACCORDING TO MICHOV ---- 180 COOMASSIE BRILLIANT BLUE R-250 STAINING OF PROTEINS RESOLVED IN SDS GELS ---- 183 REFERENCES - 184 2.8 ISOELECTRIC FOCUSING OF PROTEINS - 187 2.8.1 THEORY OF ISOELECTRIC FOCUSING ---- 188 2.8.2 ISOELECTRIC FOCUSING WITH CARRIER AMPHOLYTES ---- 189 2.8.2.1 PROPERTIES OF CARRIER AMPHOLYTES - 189 2.8.2.2 FORMATION OF A PH GRADIENT BY CARRIER AMPHOLYTES ---- 190 SEPARATOR ELECTROFOCUSING - 191 2.8.2.3 IEF WITH CARRIER AMPHOLYTES ON POLYACRYLAMIDE GELS ----- 191 THIN AND ULTRATHIN POLYACRYLAMIDE GELS FOR IEF WITH CARRIER AMPHOLYTES - 191 REHYDRATABLE POLYACRYLAMIDE GELS FOR IEF WITH CARRIER AMPHOLYTES ---- 191 2.8.2.4 SAMPLE PREPARATION AND APPLICATION ---- 192 2.8.2.5 ELECTRODE SOLUTIONS ----- 192 2.8.2.6 RUNNING ISOELECTRIC FOCUSING WITH CARRIER AMPHOLYTES ----- 193 2.8.3 ISOELECTRIC FOCUSING IN IMMOBILIZED PH GRADIENTS ---- 195 2.8.3.1 PROPERTIES OF IMMOBILINES ---- 196 2.8.3.2 CASTING IPG GELS ---- 197 REHYDRATABLE IPG GELS - 198 XVIII - - YY CONTENTS 2.8.3.3 2.8.4 2.8.5 RUNNING ISOELECTRIC FOCUSING ON IPG GELS - 198 ISOELECTRIC FOCUSING IN THE CLINICAL LABORATORY - 199 PROTOCOLS ---- 199 CASTING GELS FOR ISOELECTRIC FOCUSING WITH CARRIER AMPHOLYTES - 199 RUNNING ISOELECTRIC FOCUSING WITH CARRIER AMPHOLYTES ---- 200 CASTING IMMOBILINE GELS ---- 202 IEF WITH IPG GEL STRIPS ---- 202 REFERENCES - 206 2.9 FREE-FLOW ELECTROPHORESIS OF PROTEINS - 209 2.9.1 THEORY OF FREE-FLOW ELECTROPHORESIS ---- 209 2.9.2 TYPES OF FREE-FLOW ELECTROPHORESIS ---- 210 2.9.2.1 FREE-FLOW ZONE ELECTROPHORESIS ------ 211 2.9.2.2 FREE-FLOW ISOTACHOPHORESIS ------ 211 2.9.2.3 FREE-FLOW ISOELECTRIC FOCUSING ------ 212 2.9.3 DEVICE TECHNOLOGY OF FREE-FLOW ELECTROPHORESIS - 213 2.9.4 DETECTION SYSTEM OF FREE-FLOW ELECTROPHORESIS ----- 213 2.9.5 APPLICATIONS OF FREE-FLOW ELECTROPHORESIS ---- 214 2.9.6 PROTOCOLS ---- 214 FREE-FLOW ZONE ELECTROPHORESIS OF HUMAN T AND B LYMPHOCYTES ---- 214 REFERENCES ---- 215 2.10 CAPILLARY ELECTROPHORESIS OF PROTEINS - 217 2.10.1 THEORY OF CAPILLARY ELECTROPHORESIS OF PROTEINS - 217 2.10.2 INSTRUMENTATION ---- 218 2.10.2.1 COATING THE CAPILLARIES ---- 219 2.10.2.2 SIEVING MATRIX IN CAPILLARY ELECTROPHORESIS ---- 220 2.10.3 PRACTICE OF CAPILLARY ELECTROPHORESIS ----- 222 2.10.3.1 INJECTION ----- 222 2.10.3.2 SEPARATION ----- 222 2.10.3.3 DETECTION ----- 223 2.10.4 TYPES OF CAPILLARY ELECTROPHORESIS ----- 224 2.10.5 APPLICATIONS OF CAPILLARY ELECTROPHORESIS - 224 2.10.5.1 SERUM PROTEIN ANALYSIS - 224 2.10.5.2 HEMOGLOBINS ---- 225 2.10.5.3 ISOENZYMES ---- 226 2.10.5.4 IMMUNE COMPLEXES ----- 226 2.10.5.5 SINGLE CELL ANALYSIS ----- 227 2.10.6 PROTOCOLS ----- 227 CAPILLARY IEF OF PROTEINS ---- 227 REFERENCES ---- 228 CONTENTS - XIX 2.11 TWO-DIMENSIONAL ELECTROPHORESIS----- 231 2.11.1 THEORY OF 2D ELECTROPHORESIS ---- 231 2.11.2 ISOELECTRIC FOCUSING IN THE FIRST DIMENSION ---- 232 2.11.2.1 SAMPLE PREPARATION ---- 232 2.11.2.2 ISO-DALT AND IPG-DALT ---- 233 2.11.3 SDS DISC-ELECTROPHORESIS IN THE SECOND DIMENSION ---- 234 2.11.4 DETECTION AND EVALUATION OF PROTEINS IN 2D PHEROGRAMS ---- 236 2.11.4.1 AUTORADIOGRAPHY AND FLUOROGRAPHY ----- 236 2.11.4.2 TWO-DIMENSIONAL GEL IMAGE ANALYSIS ----- 236 2.11.5 PROTOCOLS ---- 237 TWO-DIMENSIONAL GEL ELECTROPHORESIS USING THE O ' FARRELL SYSTEM ---- 237 SAMPLE PREPARATION ---- 237 FIRST-DIMENSIONAL GELS (ISOELECTRIC FOCUSING) ---- 237 SECOND-DIMENSIONAL GELS (SDS ELECTROPHORESIS) ---- 238 REFERENCES - 239 2.12 PREPARATIVE ELECTROPHORESIS OF PROTEINS - 241 2.12.1 PREPARATIVE DISC-ELECTROPHORESIS ---- 241 2.12.1.1 ELUTION OF PROTEINS DURING ELECTROPHORESIS ---- 241 2.12.1.2 ELUTION OF PROTEINS AFTER ELECTROPHORESIS ---- 242 ELUTION BY DIFFUSION ---- 243 ELUTION BY GEL DISSOLVING ---- 243 ELECTROELUTION ---- 243 2.12.2 PREPARATIVE ISOELECTRIC FOCUSING ---- 245 2.12.2.1 PREPARATIVE IEF WITH CARRIER AMPHOLYTES IN GRANULATED GELS ---- 245 2.12.2.2 PREPARATIVE IEF IN IMMOBILIZED PH GRADIENTS ----- 247 2.12.2.3 RECYCLING ISOELECTRIC FOCUSING ---- 248 2.12.3 QPNC-PAGE ---- 248 2.12.4 PROTOCOLS ----- 249 QPNC-PAGE ---- 249 REFERENCES ---- 250 2.13 MICROCHIP ELECTROPHORESIS OF PROTEINS ---- 253 2.13.1 MICROCHIP MATERIALS 254 2.13.1.1 PDMS 254 2.13.1.2 PMMA ----- 255 2.13.1.3 PC ---- 255 2.13.2 MICROCHIP FABRICATION ------ 256 2.13.2.1 FABRICATION OF CHANNEL PLATE ---- 257 2.13.2.2 FABRICATION OF COVER PLATE ----- 258 2.13.2.3 WALL COATING ---- 258 XX - CONTENTS DYNAMIC WALL COATING ---- 258 PERMANENT WALL COATING ---- 259 2.13.2.4 BONDING THE PLATES ----- 259 2.13.3 ZONE ELECTROPHORESIS ON MICROCHIP ---- 261 2.13.3.1 FREE-FLOW ELECTROPHORESIS ON MICROCHIP ---- 261 2.13.3.2 AFFINITY AND IMMUNOELECTROPHORESIS ON MICROCHIP ---- 262 2.13.4 ISOTACHOPHORESIS ON MICROCHIP ----- 262 2.13.4.1 DISC-ELECTROPHORESIS ON MICROCHIP ----- 262 2.13.5 ISOELECTRIC FOCUSING ON MICROCHIP ----- 262 2.13.6 TWO-DIMENSIONAL ELECTROPHORESIS ON MICROCHIP - 263 2.13.7 PROTEIN SEPARATION TECHNIQUE ON MICROCHIP ---- 263 2.13.7.1 CONCENTRATING THE PROTEIN SAMPLES PRIOR TO MICROCHIP ELECTROPHORESIS ---- 263 2.13.7.2 RUNNING PROTEIN ELECTROPHORESIS ---- 264 2.13.7.3 DETECTING THE PROTEINS ---- 264 STAINING THE PROTEINS ---- 264 FLUORESCENCE DETECTION ---- 264 CHEMILUMINESCENCE DETECTION ---- 265 MASS SPECTROMETRY DETECTION ---- 265 2.13.8 MICROCHIPS IN CLINICAL DIAGNOSTICS ---- 265 REFERENCES ---- 266 2.14 BLOTTING OF PROTEINS ---- 271 2.14.1 THEORY OF PROTEIN BLOTTING ---- 271 2.14.2 BLOT MEMBRANES ---- 271 2.14.3 TRANSFER OF PROTEINS ---- 272 2.14.3.1 ELECTROTRANSFER OF PROTEINS ---- 272 TANK BLOTTING OF PROTEINS ---- 273 SEMIDRY BLOTTING OF PROTEINS ---- 273 2.14.3.2 CAPILLARY TRANSFER OF PROTEINS ---- 274 2.14.4 BLOCKING ---- 275 2.14.5 DETECTION ---- 276 2.14.5.1 DETECTION BY DYES ---- 276 2.14.5.2 DETECTION BY PROBES ---- 276 2.14.5.3 IMMUNOBLOTTING ---- 277 2.14.6 MAKING THE BLOT MEMBRANES TRANSPARENT ---- 277 2.14.7 BLOTTING TECHNIQUES ---- 278 2.14.7.1 WESTERN BLOTTING ---- 278 2.14.7.2 FAR-WESTERN BLOTTING ---- 279 2.14.7.3 SOUTHWESTERN BLOTTING ---- 279 2.14.7.4 NORTHWESTERN BLOTTING ---- 279 2.14.7.5 EASTERN BLOTTING ---- 280 CONTENTS - XXI 2.14.8 PROTOCOLS ---- 280 WESTERN BLOTTING ---- 280 SEMIDRY BLOTTING ---- 282 CAPILLARY BLOTTING ---- 283 INDIA INK STAINING OF PROTEINS ON MEMBRANE ---- 284 DETECTING PROTEINS BY IMMUNOBLOTTING ---- 285 IMMUNOPROBING WITH AVIDIN-BIOTIN COUPLING TO SECONDARY ANTIBODY ---- 286 REFERENCES - 287 2.15 EVALUATION OF PROTEIN PHEROGRAMS - 289 2.15.1 QUALITATIVE EVALUATION OF PROTEIN PHEROGRAMS - 289 2.15.1.1 FIXING OF PROTEINS ---- 290 2.15.1.2 STAINING OF PROTEINS ---- 290 ANIONIC DYE STAINING OF PROTEINS ---- 291 PONCEAU S STAINING OF PROTEINS ---- 291 AMIDO BLACK STAINING OF PROTEINS - 291 COOMASSIE BRILLIANT BLUE STAINING OF PROTEINS ---- 292 CONVENTIONAL COOMASSIE BRILLIANT BLUE STAINING OF PROTEINS ---- 292 COLLOIDAL COOMASSIE BRILLIANT BLUE STAINING OF PROTEINS ---- 293 INDIA INK STAINING OF PROTEINS ---- 293 COUNTER-IONIC DYE STAINING OF PROTEINS ---- 293 METAL STAINING OF PROTEINS - 294 SILVER STAINING OF PROTEINS - 294 CHELATE DYE STAINING OF PROTEINS ---- 295 STAINING OF GLYCOPROTEINS ---- 295 STAINING OF LIPOPROTEINS ---- 296 STAINING OF ENZYMES ---- 296 AUTORADIOGRAPHY OF PROTEINS ---- 297 FLUOROGRAPHY OF PROTEINS ---- 297 SYPRO RUBY STAINING OF PROTEINS ---- 298 DESTAINING OF GEL BACKGROUND ---- 298 DRYING OF GELS ---- 299 2.15.2 QUANTITATIVE EVALUATION OF A PROTEIN PHEROGRAM ---- 299 2.15.2.1 DENSITOMETRY ---- 299 DENSITOMETERS ---- 301 2.15.2.2 SCANNING ---- 302 2.15.3 PROTOCOLS ---- 302 COOMASSIE BRILLIANT BLUE STAINING OF PROTEINS IN POLYACRYLAMIDE GELS ---- 302 COLLOIDAL COOMASSIE STAINING ---- 303 RAPID SILVER STAINING ---- 304 XXII - CONTENTS SYPRO RUBY STAINING OF PROTEINS ---- 305 STAINING OF LIPOPROTEINS WITH SUDAN BLACK B ---- 305 REFERENCES ---- 306 2.16 PRECAST GELS FOR PROTEIN ELECTROPHORESIS. REHYDRATABLE GELS - 309 2.16.1 PRECAST AGAROSE GELS ---- 309 2.16.2 PRECAST POLYACRYLAMIDE GELS - 309 2.16.3 REHYDRATABLE POLYACRYLAMIDE GELS ---- 311 2.16.4 PROTOCOLS ---- 311 SILANIZATION OF GLASS PLATES - 311 CASTING AGAROSE GELS ON SUPPORT FILM BY CAPILLARY TECHNIQUE ---- 311 CASTING THIN PAA GELS BY CASSETTE TECHNIQUE ---- 312 CASTING ULTRATHIN PAA GELS BY FLAP TECHNIQUE ---- 314 REFERENCES - 315 3 ELECTROPHORESIS OF NUCLEIC ACIDS - 317 BUFFERS FOR ELECTROPHORESIS OF NUCLEIC ACIDS ---- 317 POLYMERASE CHAIN REACTION ---- 318 PROCEDURE ---- 318 APPLICATIONS ---- 320 SURFACE ELECTROPHORESIS ---- 321 REFERENCES ---- 323 3.1 AGAROSE GEL ELECTROPHORESIS OF NUCLEIC ACIDS - 325 3.1.1 ZONE POLYACRYLAMIDE GEL ELECTROPHORESIS OF NATIVE NUCLEIC ACIDS ---- 326 3.1.1.1 THEORY OF SIEVING MIGRATION ---- 326 FACTORS AFFECTING MIGRATION OF NUCLEIC ACIDS ---- 326 3.1.1.2 PRACTICE OF ZONE AGAROSE GEL ELECTROPHORESIS OF NATIVE NUCLEIC ACIDS ---- 327 SUBMARINE ELECTROPHORESIS OF NUCLEIC ACIDS ---- 327 RESTRICTION FRAGMENT LENGTH POLYMORPHISM ---- 328 VARIABLE NUMBER TANDEM REPEAT ---- 329 DNA PROFILING ---- 329 PATERNITY OR MATERNITY TESTING FOR CHILD ---- 330 INTERPRETATION OF DNA TEST RESULTS ---- 331 3.1.2 ZONE AGAROSE GEL ELECTROPHORESIS OF DENATURED NUCLEIC ACIDS - 332 3.1.3 DNA SEQUENCING ---- 332 3.1.3.1 MAXAM-GILBERT SEQUENCING METHOD ---- 332 3.1.3.2 SANGER SEQUENCING METHOD ----- 333 3.1.3.3 SINGLE-STRAND CONFORMATION POLYMORPHISM METHOD ---- 334 3.1.3.4 DNASE FOOTPRINTING ASSAY ----- 336 CONTENTS - - XXIII 3.1.3.5 NUCLEASE PROTECTION ASSAY ---- 337 SJ-NUDEASE PROTECTION ASSAY ---- 337 3.1.4 RNA SEPARATION ---- 338 3.1.4.1 PRIMER EXTENSION ASSAY ----- 339 3.1.5 PROTOCOLS ---- 339 NATIVE DNA ELECTROPHORESIS ON AGAROSE GELS ---- 339 SANGER SEQUENCING REACTIONS USING TAQ DNA POLYMERASE ---- 340 REFERENCES - 341 3.2 PULSED-FIELD GEL ELECTROPHORESIS OF NUCLEIC ACIDS - 343 3.2.1 THEORY OF PULSED-FIELD GEL ELECTROPHORESIS OF NUCLEIC ACIDS ---- 343 3.2.2 TYPES OF PULSED-FIELD GEL ELECTROPHORESIS ---- 344 3.2.3 MAPPING THE HUMAN GENOME ---- 345 3.2.3.1 STR ANALYSIS ---- 345 3.2.3.2 AMPFLP ---- 345 3.2.3.3 DNA FAMILY RELATIONSHIP ANALYSIS ----- 346 3.2.3.4 Y-CHROMOSOME ANALYSIS ----- 346 3.2.3.5 MITOCHONDRIAL ANALYSIS ----- 346 3.2.4 PROTOCOLS ----- 346 PULSED-FIELD GEL ELECTROPHORESIS OF CHROMOSOMAL DNA---- 346 REFERENCES ---- 347 3.3 CAPILLARY ELECTROPHORESIS OF NUCLEIC ACIDS - 349 3.3.1 THEORY OF CAPILLARY ELECTROPHORESIS OF NUCLEIC ACIDS ---- 349 3.3.2 INSTRUMENTATION FOR CAPILLARY ELECTROPHORESIS ---- 350 3.3. 2.1 CAPILLARIES ----- 350 3.3.2.2 COATING POLYMERS ----- 350 3.3.2.3 GELS ----- 351 3.3.3 RUNNING CAPILLARY ELECTROPHORESIS - 352 3.3.3.1 INJECTION ---- 352 3.3.3.2 SEPARATION ----- 352 3.3.3.3 DETECTION ----- 352 3.3.4 PULSED-FIELD CAPILLARY ELECTROPHORESIS ---- 353 3.3.5 APPLICATIONS OF CAPILLARY ELECTROPHORESIS ---- 353 3.3.6 PROTOCOLS ---- 354 QUANTITATIVE PCR ANALYSIS ---- 354 REFERENCES - 354 3.4 POLYACRYLAMIDE GEL ELECTROPHORESIS OF NUCLEIC ACIDS - 357 3.4.1 ZONE POLYACRYLAMIDE GEL ELECTROPHORESIS OF NATIVE NUCLEIC ACIDS ----- 357 XXIV - CONTENTS 3.4.1.1 PRACTICE OF ZONE POLYACRYLAMIDE GEL ELECTROPHORESIS OF NATIVE NUCLEIC ACIDS ---- 358 3.4.2 DISC-ELECTROPHORESIS OF NATIVE NUCLEIC ACIDS ----- 360 3.4.2.1 THEORY OF DISC-ELECTROPHORESIS OF NATIVE NUCLEIC ACIDS ----- 360 3.4.2.2 RUNNING DISC-ELECTROPHORESIS OF NATIVE NUCLEIC ACIDS - 360 3.4.2.3 DISC-ELECTROPHORESIS OF DOUBLE-STRANDED PCR PRODUCTS ----- 361 3.4.3 ELECTROPHORETIC MOBILITY SHIFT ASSAY ---- 363 3.4.4 CLINICAL APPLICATIONS ---- 364 3.4.5 PROTOCOLS ---- 364 DNA DISC-ELECTROPHORESIS IN TRIS-TAURINATE BUFFER AT TWO PH VALUES ACCORDING TO MICHOV ---- 364 SEPARATION OF PCR PRODUCTS BY POLYACRYLAMIDE GEL DISC ELECTROPHORESIS ACCORDING TO MICHOV ---- 366 ELECTROPHORETIC MOBILITY SHIFT ASSAY ---- 367 ELECTROELUTION OF SMALL DNA FRAGMENTS FROM POLYACRYLAMIDE GEL ---- 368 REFERENCES ---- 368 3.5 MICROCHIP ELECTROPHORESIS OF NUCLEIC ACIDS - 371 3.5.1 THEORY OF MICROCHIP ELECTROPHORESIS OF NUCLEIC ACIDS ---- 371 3.5.2 CONSTRUCTION OF A MICROCHIP FOR ELECTROPHORESIS OF NUCLEIC ACIDS ---- 371 3.5.2.1 POLYMERS ----- 372 3.5.3 RUNNING MICROCHIP ELECTROPHORESIS OF NUCLEIC ACIDS ---- 373 3.5.4 APPLICATIONS OF DNA MICROCHIP ELECTROPHORESIS ---- 373 REFERENCES ---- 373 3.6 BLOTTING OF NUCLEIC ACIDS ---- 375 3.6.1 BLOTTING PRINCIPLES ---- 375 3.6.1.1 TRANSFER ---- 375 DIFFUSION TRANSFER --- 376 CAPILLARY TRANSFER ---- 376 VACUUM TRANSFER ---- 376 ELECTROTRANSFER ---- 377 3.6.1.2 BLOCKING ----- 378 3.6.1.3 DETECTION BY PROBES, DYES, AND AUTORADIOGRAPHY ----- 378 3.6.2 SOUTHERN BLOTTING ------- 379 3.6.3 NORTHERN BLOTTING ------- 380 3.6.4 MIDDLE-EASTERN BLOTTING ---- 381 3.6.5 PROTOCOLS ---- 382 SOUTHERN BLOTTING BY DOWNWARD CAPILLARY TRANSFER ---- 382 ELECTROBLOTTING OF POLYACRYLAMIDE GEL ONTO NYLON MEMBRANE ---- 383 REFERENCES ---- 384 CONTENTS - - XXV 3.7 EVALUATION OF NUCLEIC ACID PHEROGRAMS ---- 385 3.7.1 COUNTER-ION DYE STAINING OF NUCLEIC ACIDS ---- 385 3.7.2 SILVER STAINING OF NUCLEIC ACIDS ---- 385 3.7.3 FLUORESCENCE METHODS FOR DETECTING NUCLEIC ACIDS 386 3.7.4 AUTORADIOGRAPHY OF NUCLEIC ACIDS --- 389 3.7.5 LABELING OF NUCLEIC ACIDS WITH PROTEINS ---- 390 3.7.6 ABSORPTION SPECTROSCOPY OF NUCLEIC ACIDS ----- 391 3.7.7 PROTOCOLS ---- 391 DETECTION OF DNA AND RNA IN GEL USING ETHIDIUM BROMIDE ---- 391 FAST AND SENSITIVE SILVER STAINING OF DNA BANDS ACCORDING TO HAN ETAL. [34] ---- 392 AUTORADIOGRAPHY OF RADIOLABELED DNA IN GELS AND BLOTS ---- 392 REFERENCES - 394 3.8 PRECAST GELS FOR NUCLEIC ACID ELECTROPHORESIS - 395 3.8.1 PRECAST AGAROSE GELS ---- 395 3.8.2 PRECAST POLYACRYLAMIDE GELS ---- 395 3.8.3 PROTOCOLS ---- 396 CASTING MINI AND MIDI-AGAROSE GELS ---- 396 4 IONTOPHORESIS --- 397 4.1 THEORY OF IONTOPHORESIS ---- 397 4.2 FACTORS AFFECTING IONTOPHORESIS ---- 398 4.2.1 PHYSICOCHEMICAL FACTORS ---- 398 4.2.2 BIOLOGICAL FACTORS ---- 399 4.3 CALCULATING THE IONTOPHORETIC CURRENT ---- 399 4.4 IONTOPHORESIS DEVICE ---- 399 4.5 DIAGNOSTIC IONTOPHORESIS ---- 400 4.6 THERAPEUTIC IONTOPHORESIS ---- 400 4.6.1 TRANSDERMAL IONTOPHORESIS ---- 400 HYPERHIDROSIS IONTOPHORESIS ---- 401 OTHER TRANSDERMAL APPLICATIONS - 401 4.6.2 OCULAR IONTOPHORESIS ---- 402 REFERENCES ---- 403 5 HISTORY OF ELECTROPHORESIS AND IONTOPHORESIS---- 405 5.1 HISTORY OF ELECTROPHORESIS ---- 405 5.1.1 DISCOVERY OF ELECTROPHORESIS ---- 405 5.1.2 ZONE ELECTROPHORESIS OF TISELIUS ---- 406 5.1.3 PAPER AND CELLULOSE ACETATE ELECTROPHORESIS ---- 406 5.1.4 GEL ELECTROPHORESIS ---- 406 AGAROSE GEL ELECTROPHORESIS - 406 XXVI - CONTENTS POLYACRYALAMIDE GEL ELECTROPHORESIS - 407 5.1.5 5.1.6 5.1.7 ISOELECTRIC FOCUSING - 407 TWO-DIMENSIONAL ELECTROPHORESIS ---- 407 BLOTTING ---- 408 BLOTTING OF PROTEINS - 408 BLOTTING OF NUCLEIC ACIDS ---- 409 5.1.8 5.1.9 5.2 5.2.1 STAINING METHODS ---- 409 OUTLINE HISTORY OF ELECTROPHORESIS - 409 HISTORY OF IONTOPHORESIS ---- 413 OUTLINE HISTORY OF IONTOPHORESIS - 414 REFERENCES ---- 415 6 6.1 6.2 6.3 6.4 6.5 TROUBLESHOOTING ---- 421 PROTEIN ELECTROPHORESIS TROUBLESHOOTING ---- 421 IEF TROUBLESHOOTING ---- 421 NUCLEIC ACID ELECTROPHORESIS TROUBLESHOOTING - 427 BLOTTING TROUBLESHOOTING - 431 IONTOPHORESIS TROUBLESHOOTING - 433 PROBLEMS ---- 435 1 FUNDAMENTALS OF ELECTROPHORESIS ----- 435 2 ELECTROPHORESIS OF PROTEINS ---- 436 3 ELECTROPHORESIS OF NUCLEIC ACIDS ----- 438 SOLUTION OF PROBLEMS - 441 1 FUNDAMENTALS OF ELECTROPHORESIS - 441 2 ELECTROPHORESIS OF PROTEINS ---- 442 3 ELECTROPHORESIS OF NUCLEIC ACIDS ---- 445 REAGENTS FOR ELECTROPHORESIS - 447 RECIPES FOR ELECTROPHORESIS SOLUTIONS - 459 BUFFERS ---- 459 SOLUTIONS FOR AGAROSE GEL ELECTROPHORESIS ---- 463 SOLUTIONS FOR AFFINITY ELECTROPHORESIS ---- 465 SOLUTIONS FOR NATIVE DISC-ELECTROPHORESIS ---- 466 SOLUTIONS FOR SDS DISC-ELECTROPHORESIS ---- 467 SOLUTIONS FOR IEF ---- 468 BLOTTING SOLUTIONS ---- 469 FIXATIVE SOLUTIONS ---- 470 STAINING SOLUTIONS ---- 471 DESTAINING SOLUTIONS ---- 472 CONTENTS - - XXVII SILVER STAINING SOLUTIONS ---- 472 OTHER SOLUTIONS ---- 473 SI UNITS AND PHYSICAL CONSTANTS USED IN ELECTROPHORESIS - 475 REFERENCES ---- 479 ELECTROPHORESIS TERMS - 481 INDEX ---- 485
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isbn	3110761629 9783110761627
language	English
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publisher	De Gruyter
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spelling	Michov, Budin 1944- Verfasser (DE-588)1066981523 aut Electrophoresis fundamentals essential theory and practice Budin Michov Berlin ; Boston De Gruyter [2022] © 2022 XXXI, 490 Seiten Illustrationen, Diagramme txt rdacontent n rdamedia nc rdacarrier De Gruyter graduate Elektrophorese (DE-588)4014373-9 gnd rswk-swf Analytical Chemistry Biochemie Biochemistry Chemistry Electrophorese Electrophoresis Laboratory Medicine. Molekularbiologie Molekulare Medizin TB: Textbook Elektrophorese (DE-588)4014373-9 s DE-604 Walter de Gruyter GmbH & Co. KG (DE-588)10095502-2 pbl Erscheint auch als Online-Ausgabe, PDF 978-3-11-076164-1 Erscheint auch als Online-Ausgabe, EPUB 978-3-11-076167-2 X:MVB https://www.degruyter.com/isbn/9783110761627 DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=033282207&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis
spellingShingle	Michov, Budin 1944- Electrophoresis fundamentals essential theory and practice Elektrophorese (DE-588)4014373-9 gnd
subject_GND	(DE-588)4014373-9
title	Electrophoresis fundamentals essential theory and practice
title_auth	Electrophoresis fundamentals essential theory and practice
title_exact_search	Electrophoresis fundamentals essential theory and practice
title_exact_search_txtP	Electrophoresis fundamentals essential theory and practice
title_full	Electrophoresis fundamentals essential theory and practice Budin Michov
title_fullStr	Electrophoresis fundamentals essential theory and practice Budin Michov
title_full_unstemmed	Electrophoresis fundamentals essential theory and practice Budin Michov
title_short	Electrophoresis fundamentals
title_sort	electrophoresis fundamentals essential theory and practice
title_sub	essential theory and practice
topic	Elektrophorese (DE-588)4014373-9 gnd
topic_facet	Elektrophorese
url	https://www.degruyter.com/isbn/9783110761627 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=033282207&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA
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