Introduction |
|
v | |
Part I Isoelectric Focussing: Fundamentals. Perspectives and Limits. Optimization of the Separation Process |
|
|
|
3 | (6) |
Part I.I Isoelectric Focussing: Fundamentals |
|
|
Electrolyte Dissociation in Water Solution. Simple Electrolytes |
|
|
9 | (14) |
|
|
9 | (1) |
|
Stepwise and parallel dissociation schemes for a bivalent protolyte |
|
|
9 | (1) |
|
Relative concentration of different protolyte forms for stepwise and parallel schemes |
|
|
10 | (2) |
|
Hydrogen ions concentration and buffer capacity |
|
|
12 | (2) |
|
|
14 | (2) |
|
|
16 | (1) |
|
Mobility of protolyte molecule |
|
|
16 | (1) |
|
Non-additive sum for buffer capacity in case of stepwise dissociation |
|
|
17 | (1) |
|
Non-amphoteric compounds and buffer capacity in `isoprotic state' |
|
|
18 | (2) |
|
|
20 | (1) |
|
|
21 | (2) |
|
Dissociation of Polyvalent Electrolytes |
|
|
23 | (16) |
|
|
23 | (1) |
|
Acid-base equilibria, macroscopic and microscopic constants |
|
|
24 | (3) |
|
Dissociation schemes of a hybrid type |
|
|
27 | (2) |
|
Proton transfer tautomerism |
|
|
29 | (1) |
|
Schemes with independent dissociation |
|
|
30 | (1) |
|
Titration curve modelling |
|
|
31 | (1) |
|
Linderstrøm-Lang equation |
|
|
32 | (1) |
|
Calculation of the complete set of microconstants |
|
|
32 | (2) |
|
Relative concentration of microstates for a homopolymer (independent dissociation) |
|
|
34 | (2) |
|
|
36 | (1) |
|
|
37 | (2) |
|
Kinetic Aspects of Acid-Base Equilibria |
|
|
39 | (6) |
|
|
39 | (1) |
|
Life-time of microscopic states |
|
|
40 | (1) |
|
Relaxation of the ionic atmosphere |
|
|
40 | (2) |
|
Modelling of the electrophoretic flux, electrophoretic mobility and conductivity |
|
|
42 | (2) |
|
|
44 | (1) |
|
|
45 | (10) |
|
|
45 | (1) |
|
Simplest examples of natural pH gradients |
|
|
45 | (6) |
|
pH gradients created with a multi-component mixture of atmophteric compounds |
|
|
51 | (2) |
|
|
53 | (2) |
|
|
55 | (20) |
|
Classical immobilised pH gradients created with linear density gradient |
|
|
55 | (1) |
|
Linear pH gradients with non-linear gradients of concentration |
|
|
56 | (1) |
|
Buffering and conductivity properties of immobilised pH gradients |
|
|
57 | (4) |
|
Some characteristic features of electrophoresis in gel media with immobilised electric charge |
|
|
61 | (12) |
|
Method of diagonal sample application |
|
|
61 | (1) |
|
Experimentally observed dynamics of isoelectric focussing in immobilised pH gradient gels |
|
|
62 | (4) |
|
Low-molecular mass ion adsorption on weak ion exchanger |
|
|
66 | (7) |
|
|
73 | (1) |
|
|
73 | (2) |
|
|
75 | (6) |
|
|
75 | (1) |
|
Steady-state concentration distribution with an assumption of no sample-buffer interaction |
|
|
75 | (1) |
|
The influence of the focussing sample on gradient properties |
|
|
76 | (4) |
|
Low sample concentrations |
|
|
76 | (3) |
|
High sample concentrations |
|
|
79 | (1) |
|
|
80 | (1) |
|
The Dynamics of Isoelectric Focussing |
|
|
81 | (6) |
|
|
81 | (1) |
|
Diffusionless approximation |
|
|
81 | (2) |
|
The evaluation of focussing time |
|
|
83 | (1) |
|
|
84 | (3) |
Part I.II Optimization of the Electrophoretic Separation |
|
|
|
87 | (8) |
|
|
87 | (1) |
|
Buffer capacity and buffer resource |
|
|
87 | (2) |
|
Buffer properties of solutions of proteins and nucleic acids |
|
|
89 | (3) |
|
Biopolymers as titration agents |
|
|
92 | (1) |
|
|
93 | (2) |
|
Optimisation of Electrophoretic Separation |
|
|
95 | (10) |
|
Optimisation of electrophoretic separation using pH-charge relationship |
|
|
95 | (6) |
|
Calculation of mobility vs. pH |
|
|
95 | (1) |
|
Relative charge difference for two components to be separated |
|
|
96 | (5) |
|
Dependence of mobility on molecular mass in free solution |
|
|
101 | (2) |
|
Isoelectric buffers. The concept of `normalised β/λ ratio' |
|
|
103 | (1) |
|
|
104 | (1) |
|
|
105 | (4) |
|
Two-dimensional electrophoresis |
|
|
105 | (1) |
|
Other two-dimensional separations |
|
|
106 | (1) |
|
Mobility versus pH curves |
|
|
107 | (1) |
|
|
108 | (1) |
|
Limitations of the Method of Isoelectric Focussing |
|
|
109 | (14) |
|
|
109 | (1) |
|
Ways of generating pH gradients |
|
|
110 | (4) |
|
Natural and artificial pH gradients |
|
|
110 | (1) |
|
|
110 | (1) |
|
External temperature field |
|
|
110 | (3) |
|
Thermal pH gradients created by Joule heat dissipation in an electrophoretic chamber with a non-constant cross-section |
|
|
113 | (1) |
|
Gradient in dielectric constant |
|
|
113 | (1) |
|
Dielectric constant influence on buffer dissociation constant |
|
|
113 | (1) |
|
Gradient of electric field coupled with dielectric constant gradient |
|
|
114 | (1) |
|
|
114 | (2) |
|
Microheterogeneity of proteins and other biopolymers |
|
|
116 | (2) |
|
pH shift due to single modifications of ionogenic groups |
|
|
116 | (1) |
|
Multiple one-type modifications |
|
|
117 | (1) |
|
|
118 | (5) |
Part II Methodology |
|
|
Conventional Isoelectric Focussing in Gel Slabs and Capillaries and Immobilised pH Gradients |
|
|
123 | (94) |
|
|
124 | (3) |
|
A brief historical survey |
|
|
125 | (2) |
|
Conventional isoelectric focussing in amphoteric buffers |
|
|
127 | (39) |
|
|
127 | (1) |
|
|
128 | (1) |
|
Applications and limitations |
|
|
129 | (1) |
|
|
130 | (1) |
|
|
130 | (2) |
|
|
132 | (1) |
|
Electrophoretic equipment |
|
|
132 | (1) |
|
|
132 | (1) |
|
|
132 | (1) |
|
|
132 | (1) |
|
|
133 | (1) |
|
|
133 | (1) |
|
|
134 | (1) |
|
|
135 | (1) |
|
|
135 | (1) |
|
The Polyacrylamide gel matrix |
|
|
136 | (1) |
|
|
136 | (2) |
|
|
138 | (1) |
|
Choice of carrier ampholytes |
|
|
138 | (3) |
|
Gel preparation and electrophoresis |
|
|
141 | (1) |
|
|
142 | (1) |
|
|
142 | (2) |
|
|
144 | (1) |
|
Sample loading and electrophoresis |
|
|
144 | (4) |
|
|
148 | (3) |
|
Micellar Coomassie Blue G-250 |
|
|
151 | (1) |
|
Coomassie Blue R-250/CuSo4 |
|
|
151 | (1) |
|
Coomassie Blue R-250/sulphosalicylic acid |
|
|
152 | (1) |
|
|
152 | (1) |
|
Coomassie Blue G-250/urea/perchloric acid |
|
|
153 | (1) |
|
Fluorescence protein detection |
|
|
153 | (1) |
|
Specific protein detection methods |
|
|
154 | (1) |
|
Quantitation of the focussed bands |
|
|
154 | (1) |
|
|
155 | (1) |
|
Waviness of bands near the anode |
|
|
155 | (1) |
|
Burning along the cathodic strip |
|
|
155 | (1) |
|
pH gradients different from expected |
|
|
155 | (1) |
|
Sample precipitation at the application point |
|
|
155 | (1) |
|
Some typical applications of IEF |
|
|
156 | (1) |
|
Examples of some fractionations |
|
|
157 | (5) |
|
|
162 | (4) |
|
|
166 | (22) |
|
|
166 | (1) |
|
The problems of conventional IEF |
|
|
166 | (1) |
|
|
167 | (3) |
|
Narrow and ultra narrow pH gradients |
|
|
170 | (4) |
|
Extended pH gradients: general rules for their generation and optimisation |
|
|
174 | (1) |
|
Non-linear, extended pH gradients |
|
|
175 | (2) |
|
Extremely alkaline pH gradients |
|
|
177 | (1) |
|
|
178 | (1) |
|
Casting an Immobiline gel |
|
|
178 | (4) |
|
Reswelling dry Immobiline gels |
|
|
182 | (1) |
|
|
183 | (1) |
|
Staining and pH measurements |
|
|
184 | (1) |
|
Storage of the Immobiline chemicals |
|
|
184 | (1) |
|
|
185 | (1) |
|
|
186 | (1) |
|
Some analytical results with IPGs |
|
|
187 | (1) |
|
Capillary isoelectric focussing (cIEF) |
|
|
188 | (9) |
|
|
188 | (2) |
|
|
190 | (1) |
|
General guidelines for cIEF |
|
|
190 | (1) |
|
Increasing the resolution by altering the slope of the pH gradient |
|
|
191 | (3) |
|
On the problem of protein solubility at their pI |
|
|
194 | (2) |
|
Assessment of pH gradients and pI values in cIEF |
|
|
196 | (1) |
|
Separation of peptides and proteins by CZE in isoelectric buffers |
|
|
197 | (10) |
|
General properties of amphoteric, isoelectric buffers |
|
|
198 | (3) |
|
Examples of some separations of proteins in isoelectric buffers |
|
|
201 | (3) |
|
Troubleshooting for CZE in isoelectric buffers |
|
|
204 | (3) |
|
|
207 | (1) |
|
|
208 | (9) |
|
Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) |
|
|
217 | (58) |
|
|
218 | (1) |
|
SDS-protein complexes: a refinement of the model |
|
|
219 | (2) |
|
Theoretical background of Mr measurement by SDS-PAGE |
|
|
221 | (4) |
|
|
225 | (17) |
|
Purity and detection of SDS |
|
|
225 | (1) |
|
|
225 | (1) |
|
Prelabelling with dyes or fluorescent markers |
|
|
226 | (2) |
|
Post-electrophoretic detection |
|
|
228 | (1) |
|
Non-diamine, silver nitrate stain |
|
|
229 | (1) |
|
|
230 | (1) |
|
|
231 | (1) |
|
Turbidimetric protein detection (negative stain) |
|
|
231 | (1) |
|
|
232 | (1) |
|
Fluorescent detection with SYPRO dyes |
|
|
233 | (2) |
|
Possible sources of artefactual protein modification |
|
|
235 | (1) |
|
On the use and properties of surfactants |
|
|
236 | (4) |
|
The use of surfactants other than SDS |
|
|
240 | (2) |
|
|
242 | (1) |
|
Gel casting and buffer systems |
|
|
242 | (19) |
|
|
243 | (2) |
|
The standard method using continuous buffers |
|
|
245 | (1) |
|
The composition of gels and buffers |
|
|
246 | (1) |
|
Use of discontinuous buffers |
|
|
247 | (2) |
|
|
249 | (1) |
|
|
250 | (1) |
|
|
251 | (4) |
|
Peptide mapping by SDS-PAGE |
|
|
255 | (3) |
|
SDS-PAGE in photopolymerised gels |
|
|
258 | (3) |
|
|
261 | (7) |
|
Capillary and electrophoretic transfer |
|
|
262 | (2) |
|
Detection systems after blotting |
|
|
264 | (4) |
|
|
268 | (1) |
|
|
269 | (6) |
|
|
275 | (104) |
|
|
276 | (4) |
|
The early days and the evolution of 2-D PAGE |
|
|
277 | (1) |
|
A glimpse at modern times |
|
|
278 | (2) |
|
Some basic methodology pertaining to 2-D PAGE |
|
|
280 | (29) |
|
Methods of cell disruption |
|
|
282 | (1) |
|
Proteolytic attack during cell disruption |
|
|
283 | (3) |
|
|
286 | (1) |
|
Removal of interfering substances |
|
|
287 | (6) |
|
|
293 | (7) |
|
|
300 | (7) |
|
Sequential sample extraction |
|
|
307 | (2) |
|
Mass spectrometry in proteomics |
|
|
309 | (20) |
|
MALDI-TOF mass spectrometry |
|
|
311 | (7) |
|
|
318 | (3) |
|
Nanoelectrospray mass spectrometry |
|
|
321 | (2) |
|
Mass spectrometry for quantitative proteomics |
|
|
323 | (1) |
|
Labelling before extraction |
|
|
324 | (1) |
|
Labelling after extraction |
|
|
325 | (2) |
|
Multidimensional chromatography coupled to mass spectrometry |
|
|
327 | (2) |
|
Informatics and proteome: interrogating databases |
|
|
329 | (22) |
|
An example of navigation on 2-D map sites |
|
|
331 | (6) |
|
|
337 | (1) |
|
TrEMBL: a supplement to SWISS-PROT |
|
|
338 | (1) |
|
The SWISS-2DPAGE database |
|
|
338 | (4) |
|
Database searching via mass-spectrometric information |
|
|
342 | (9) |
|
Pre-fractionation tools in proteome analysis |
|
|
351 | (17) |
|
Sample pre-fractionation via different chromatographic approaches |
|
|
352 | (6) |
|
Sample pre-fraction via multicompartment electrolysers with Immobiline membranes |
|
|
358 | (10) |
|
Non-denaturing protein maps |
|
|
368 | (2) |
|
|
370 | (9) |
Acknowledgements |
|
379 | (2) |
Abbreviations in Part II |
|
381 | (2) |
Subject Index |
|
383 | |