Immobilized pH gradients: theory and methodology
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| Main Author: | |
|---|---|
| Format: | Book |
| Language: | English |
| Published: |
Amsterdam u.a.
Elsevier
1990
|
| Series: | Laboratory techniques in biochemistry and molecular biology
20 |
| Subjects: | |
| Online Access: | Inhaltsverzeichnis |
| Item Description: | Literaturverz. S. [375] - 394 |
| Physical Description: | XV, 397 S. zahlr. Ill. u. graph. Darst. |
| ISBN: | 0444813152 0444813012 0720442001 |
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| 100 | 1 | |a Righetti, Pier Giorgio |e Verfasser |4 aut | |
| 245 | 1 | 0 | |a Immobilized pH gradients |b theory and methodology |c Pier Giorgio Righetti |
| 264 | 1 | |a Amsterdam u.a. |b Elsevier |c 1990 | |
| 300 | |a XV, 397 S. |b zahlr. Ill. u. graph. Darst. | ||
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Record in the Search Index
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| adam_text | IMAGE 1
CONTENTS
LIST OF ABBREVIATIONS XI
ACKNOWLEDGMENTS XV
INTRODUCTION 1
CHAPTER 1. THE CHEMICALS 5
1.1 ISOELECTRIC FOCUSING: AN EVOLVING CONCEPT 5
1.1.1 THE FIRST GENERATION 5
1.1.2 THE SECOND GENERATION 7
1.1.3 THE THIRD GENERATION 8
1.1.4 THE FOURTH GENERATION 11
1.2 COMPARISON BETWEEN CARRIER AMPHOLYTE AND IMMOBILINE CHEMICALS . . ..
13 1.3 STRUCTURE AND SYNTHESIS OF ACIDIC IMMOBILINE 15
1.4 STRUCTURE AND SYNTHESIS OF BASIC IMMOBILINE 19
1.5 PHYSICO-CHEMICAL PROPERTIES OF IMMOBILINE 23
1.6 PROBLEMS WITH THE IMMOBILINE CHEMICALS 28
1.6.1 HYDROLYSIS OF IMMOBILINE 31
1.6.2 AUTOPOLYMERIZATION 32
1.6.3 PH-DEPENDENT FORMATION OF N-OXIDES 36
1.7 STORAGE OF IMMOBILINE: A NEW GENERATION 42
1.8 CONCLUSIONS 51
CHAPTER 2. THEORY 53
2.1 PER ASPERA AD ASTRA 53
2.2 NARROW AND ULTRA-NARROW PH GRADIENTS 54
2.2.1 THE PRINCIPLE OF AN IMMOBILINE GRADIENT 54
2.2.2 THE HENDERSON-HASSELBALCH (H-H) EQUATION 55
2.2.3 NARROW AND ULTRA-NARROW PH GRADIENTS WITH MID-POINT CENTRED OR
REMOVED FROM THE BUFFERING PK 56
2.2.4 SELECTION CRITERIA FOR 1 PH UNIT WIDE IPGS: THE TANDEM PRINCIPLE .
58 2.2.5 THE USE OF NOMOGRAMS 59
2.2.6 INTERPOLATION OF ULTRA-NARROW PH GRADIENTS BASED ON THE TANDEM
PRINCIPLE 68
IMAGE 2
IMMOBILIZED PH GRADIENTS
2.3 EXTENDED PH GRADIENTS 70
2.3.1 MULTI-CHAMBER MIXERS 70
2.3.2 TWO-CHAMBER MIXERS WITH IDENTICAL BUFFER CONCENTRATION 74 2.3.3
TWO-CHAMBER MIXERS WITH DIFFERENT BUFFER CONCENTRATIONS 77 2.3.4
NON-LINEAR, EXTENDED PH GRADIENTS 85
2.3.5 COMPUTER SIMULATIONS 87
2.3.6 HOW TO SMOOTH THE /? POWER 87
2.3.7 HOW TO CHOOSE THE TITRANTS _ 90
2.3.8 ON THE ARCHITECTURE OF OUR COMPUTER PROGRAM 90
2.3.9 THE SIMULATION AND OPTIMIZATION PROGRAM 92
2.4 HOW TO DEAL WITH EXPERIMENTAL ERRORS 96
2.5 IONIC STRENGTH ( /) 98
2.6 BUFFERING CAPACITY (/3) 99
2.7 CONDUCTIVITY 100
2.8 ELECTROENDOSMOSIS 101
2.9 THE LIMITS OF IPGS: HOW TO BREAK BARRIERS 103
2.10 RESOLVING POWER. RILBE S EQUATION 108
2.11 ALTERNATIVES FOR BUFFER FOCUSING I LL
2.12 CONCLUSIONS 115
CHAPTER 3. ANALYTICAL IPGS 117
3.1 GENERAL CONSIDERATIONS 117
3.1.1 THE BASIC METHOD 117
3.1.2 APPLICATIONS AND LIMITATIONS 118
3.1.3 SPECIFIC ADVANTAGES 119
3.2 EQUIPMENT 119
3.2.1 ELECTROPHORETIC EQUIPMENT 119
3.2.2 POLYMERIZATION CASSETTE 120
3.3 THE POLYACRYLAMIDE GEL MATRIX 124
3.3.1 REAGENTS 124
3.3.2 GEL FORMULATIONS 125
3.3.3 RECIPES FOR IPG INTERVALS 126
3.4 GEL PREPARATION AND ELECTROPHORESIS 127
3.4.1 CASTING AN IMMOBILINE GEL 127
3.4.2 RESWELLING DRY IMMOBILINE GELS 134
3.4.3 ELECTROPHORESIS 139
3.5 GENERAL EXPERIMENTAL CONSIDERATIONS 140
3.5.1 THE USE OF DENSITY AGENTS 140
3.5.2 ON THE TEMPERATURE OF POLYMERIZATION 142
3.5.3 ON THE GRADIENT MIXER 146
3.5.4 ON THE NUMBER OF WASHING STEPS 146
3.5.5 ON SAMPLE APPLICATION 149
3.5.6 FOCUSING AT PH EXTREMES 154
3.5.7 PH MEASUREMENTS 155
3.6 BLOTTING FROM IMMOBILINE GELS 158
3.7 MIXED-BED, CA-IPG GELS 160
3.8 EFFECT OF SALTS ON PROTEIN PATTERNS IN IPG GELS 165
IMAGE 3
CONTENTS
3 9 GENERAL STAINING PROCEDURES 171
3.9.1 VISIBLE ORGANIC STAINS 171
3.9.2 FLUORESCENT STAINS 173
3.9.3 SILVER STAINING 174
3.9.4 AUTORADIOGRAPHY AND FLUOROGRAPHY 175
3.10 TROUBLE SHOOTING 179
3.11 CONCLUSIONS 179
CHAPTER 4. TWO-DIMENSIONAL (2-D) MAPS 181
4.1 INTRODUCTION 181
4.2 SAMPLE PREPARATION 183
4.2.1 BODY FLUIDS 185
4.2.2 TISSUE SAMPLES 187
4.2.3 CIRCULATING AND CULTURED CELLS 187
4.2.4 PLANT TISSUES 188
4.3 SAMPLE SOLUBILIZATION 189
4.4 CHOICE OF DETERGENT 190
4.4.1 MATERIAL TO BE SOLUBILIZED 194
4.4.2 DETERGENT PROPERTIES DETRIMENTAL TO SUBSEQUENT PROCEDURES . . ..
194 4.4.3 NEW CLASSES OF DETERGENTS 196
4.5 RADIOLABELLING TECHNIQUES 198
4.5.1 RADIOIODINATION 199
4.5.2 RADIOLABELLING GLYCOPROTEINS AND GLYCOLIPIDS 201
4.5.3 BIOSYNTHETIC LABELLING 202
4.5.4 POST-SYNTHETIC LABELLING 203
4.6 THE FIRST (IPG) DIMENSION 205
4.6.1 THE GEL MATRIX 205
4.6.2 ADDITIVES 206
4.6.3 THE IPG GRADIENT. ADDITION OF CARRIER AMPHOLYTES (CA) 206 4.6.4
SAMPLE APPLICATION 207
4.6.5 FOCUSING CONDITIONS 210
4.6.6 PH GRADIENT ESTIMATION 211
4.6.7 INTERFACING THE 1ST TO THE 2ND DIMENSION 212
4.7 THE SECOND DIMENSION 216
4.7.1 THE INTERFACING SOLUTION 216
4.7.2 SOLUTIONS FOR THE 2ND-D GEL 217
4.7.3 GEL CASSETTE PREPARATION 219
4.7.4 POURING GRADIENT GELS 220
4.7.5 M T MARKERS 224
4.8 REPRODUCIBILITY OF SPOT POSITIONS IN 2-D MAPS 225
4.9 COMPUTER DATA ACQUISITION 226
4.9.1 GEL DIGITIZING AND IMAGE PROCESSING 229
4.9.2 IMAGE CLEANING 230
4.9.3 SPOT DETECTORS AND INTEGRATORS 230
4.9.4 GEL MATCHING 231
4.10 SILVER STAINING 232
4.10.1 GENERAL COMMENTS ON SILVERING 233
4.11 BLOTTING OF SPOTS FROM 2-D MAPS 237
4.11.1 IMMUNOCHEMICAL LABELLING AFTER BLOTTING 241
IMAGE 4
IMMOBILIZED PH GRADIENTS
4.12 GENERAL EXPERIMENTAL ASPECTS 245
4.13 TROUBLE SHOOTING 247
4.14 EXAMPLES OF SOME APPLICATIONS 250
4.14.1 ANALYSIS OF WHEAT ENDOSPERM AND PLANT PROTEINS 250
4.14.2 SERUM AND PLASMA PROTEINS 251
4.14.3 2-D MAPS OF URINES AND OTHER BODY FLUIDS 251
4.14.4 2-D MAPS IN GENETIC DISEASE 252
4.15 TO BE OR NOT TO BE? 254
CHAPTER 5. PREPARATIVE ASPECTS OF I M M O B I L I Z ED PH GRADIENTS . .
.. 257
5.1 INTRODUCTION 257
5.2 THEORY 259
5.2.1 PREDICTION OF ACCEPTABLE PROTEIN LOADS IN IPGS 259
5.2.2 OPTIMIZATION OF ENVIRONMENTAL PARAMETERS 262
5.2.3 GENERAL CONSIDERATIONS ON THE LOAD ABILITY AND THE IONIC STRENGTH
OF THE MILIEU 264
5.2.4 PROTEIN LOAD AS A FUNCTION OF % T IN THE MATRIX 269
5.3 METHODOLOGY 272
5.3.1 GEL CASTING 272
5.3.2 PROTEIN DETECTION 276
5.3.3 PROTEIN ELUTION 278
5.3.4 AN EXAMPLE OF A PURIFICATION PROTOCOL 289
5.4 IMMOBILINE CANAL TECHNIQUE 292
5.5 REVERSIBLE POLYACRYLAMIDE GELS 295
5.6 SEGMENTED IMMOBILIZED PH GRADIENTS 296
5.6.1 VERTICAL CHAMBERS 297
5.6.2 HORIZONTAL CHAMBERS 303
5.6.3 MEMBRANE APPARATUS 306
5.6.4 ON THE UPPER LOAD IN ELECTROKINETIC PROCESSES 311
5.7 ALTERNATIVE PREPARATIVE METHODS 311
5.7.1 THE BIOSTREAM SEPARATOR 311
5.7.2 RECYCLING ISOELECTRIC FOCUSING (RIEF) 313
5.7.3 FREE-FLOW, FIELD STEP FOCUSING 314
5.8 CONCLUSIONS 316
CHAPTER 6. SOME APPLICATIONS OF IPGS 317
6.1 ANALYSIS OF HEMOGLOBINS 317
6.1.1 DETECTION OF CHARGED VARIANTS 318
6.1.2 DETECTION OF NEUTRAL MUTATIONS 319
6.1.3 THALASSEMIA SCREENING 326
6.1.4 ANIMAL HEMOGLOBINS 330
6.1.5 LEGHEMOGLOBINS 331
6.2 POLYMORPHISM OF HUMAN PROTEINS 333
6.2.1 CTJ-ACIDIC GLYCOPROTEIN 334
6.2.2 ACID PHOSPHATASE 335
6.2.3 ALBUMIN AND PREALBUMIN 336
IMAGE 5
6.2.4 A,-ANTITRYPSIN (A RAT) 337
6.2.5 APOLIPROTEINS 339
6.2.6 C3 344
6 2 7 FACTOR B 345
6.2.8 FACTOR XIIIB 345
6.2.9 GROUP SPECIFIC COMPONENT 346
6.2.10 LECITHIN : CHOLESTEROL ACYLTRANSFERASE 348
6.2.11 PHOSPHOGLUCOMUTASE 348
6.2.12 PROTEIN C 349
6.2.13 TRANSFERRIN 350
6.3 ZYMOGRAMS 354
6.4 FOCUSING AT PH EXTREMES 361
6.5 CONCLUSIONS. ON PROTEIN HETEROGENEITY 369
CHAPTER 7. POST SCRIPTUM: MAESTRO VOLTA 373
REFERENCES 375
SUBJECT INDEX 395
|
| any_adam_object | 1 |
| author | Righetti, Pier Giorgio |
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| bvnumber | BV002577973 |
| classification_rvk | WC 3440 WC 4150 |
| classification_tum | CHE 228f CHE 802f CHE 810f CHE 145f |
| ctrlnum | (OCoLC)243418951 (DE-599)BVBBV002577973 |
| discipline | Physik Biologie Chemie |
| format | Book |
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| genre | Ph-Gradient gnd |
| genre_facet | Ph-Gradient |
| id | DE-604.BV002577973 |
| illustrated | Illustrated |
| indexdate | 2024-07-09T15:47:01Z |
| institution | BVB |
| isbn | 0444813152 0444813012 0720442001 |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-001660765 |
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| physical | XV, 397 S. zahlr. Ill. u. graph. Darst. |
| publishDate | 1990 |
| publishDateSearch | 1990 |
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| publisher | Elsevier |
| record_format | marc |
| series | Laboratory techniques in biochemistry and molecular biology |
| series2 | Laboratory techniques in biochemistry and molecular biology |
| spelling | Righetti, Pier Giorgio Verfasser aut Immobilized pH gradients theory and methodology Pier Giorgio Righetti Amsterdam u.a. Elsevier 1990 XV, 397 S. zahlr. Ill. u. graph. Darst. txt rdacontent n rdamedia nc rdacarrier Laboratory techniques in biochemistry and molecular biology 20 Literaturverz. S. [375] - 394 Isoelectric Focussing cabt Electrophoresis cabt Theorie (DE-588)4059787-8 gnd rswk-swf Immobilisierung (DE-588)4123417-0 gnd rswk-swf Methode (DE-588)4038971-6 gnd rswk-swf Wasserstoffionenkonzentration (DE-588)4189273-2 gnd rswk-swf Ph-Gradient gnd rswk-swf Ph-Gradient f Immobilisierung (DE-588)4123417-0 s DE-604 Wasserstoffionenkonzentration (DE-588)4189273-2 s Methode (DE-588)4038971-6 s Theorie (DE-588)4059787-8 s Laboratory techniques in biochemistry and molecular biology 20 (DE-604)BV005871299 20 SWB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=001660765&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Righetti, Pier Giorgio Immobilized pH gradients theory and methodology Laboratory techniques in biochemistry and molecular biology Isoelectric Focussing cabt Electrophoresis cabt Theorie (DE-588)4059787-8 gnd Immobilisierung (DE-588)4123417-0 gnd Methode (DE-588)4038971-6 gnd Wasserstoffionenkonzentration (DE-588)4189273-2 gnd |
| subject_GND | (DE-588)4059787-8 (DE-588)4123417-0 (DE-588)4038971-6 (DE-588)4189273-2 |
| title | Immobilized pH gradients theory and methodology |
| title_auth | Immobilized pH gradients theory and methodology |
| title_exact_search | Immobilized pH gradients theory and methodology |
| title_full | Immobilized pH gradients theory and methodology Pier Giorgio Righetti |
| title_fullStr | Immobilized pH gradients theory and methodology Pier Giorgio Righetti |
| title_full_unstemmed | Immobilized pH gradients theory and methodology Pier Giorgio Righetti |
| title_short | Immobilized pH gradients |
| title_sort | immobilized ph gradients theory and methodology |
| title_sub | theory and methodology |
| topic | Isoelectric Focussing cabt Electrophoresis cabt Theorie (DE-588)4059787-8 gnd Immobilisierung (DE-588)4123417-0 gnd Methode (DE-588)4038971-6 gnd Wasserstoffionenkonzentration (DE-588)4189273-2 gnd |
| topic_facet | Isoelectric Focussing Electrophoresis Theorie Immobilisierung Methode Wasserstoffionenkonzentration Ph-Gradient |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=001660765&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| volume_link | (DE-604)BV005871299 |
| work_keys_str_mv | AT righettipiergiorgio immobilizedphgradientstheoryandmethodology |