Electrophoresis in Practice, 3rd Edition

by Reiner Westermeier (Amersham Pharmacia biotech Europe GmbH Freiburg, Germany)

• ISBN13: 9783527303007
• ISBN10: 3527303006
• Format: Hardcover
• Copyright: 2001-01-01
• Publisher: Wiley-VCH

Summary

This is your "Laboratory Guide" for successful electrophoretic separations ! Organized in two parts it gives the reader a thorough presentation of the fundamentals followed by a detailed description of 15 of the most common methods currently in use. The Third Edition now includes the latest developments in 2D-electrophoresis as well as an overview of the proteome analysis methodology. From reviews of the previous editions: "...The rigorous description of each method along with the extensive figures will easily allow the novice to reproduce these methods in the laboratory..." --The Analyst "...Perhaps the most important point about the book is that all the recipes provided do actually work.... --Journal of Laboratory Medicine "...an excellent book which we recommend greatly and which has not to be missed in any laboratory of cellular and molecular biology which respects it self!" --Cellular and Molecular Biology "...As a comprehensive guide to the huge variety of electrophoretic methods now available, this is very good value...The superb...troubleshooting appendix almost justifies the price on ist own..." --Laboratory Equipment Digest

Table of Contents

Part I Fundamentals
Introduction	1	(6)
Electrophoresis	7	(34)
General	7	(8)
Electrophoresis in non-restrictive gels	15	(4)
Agarose gel electrophoresis	15	(3)
Polyacrylamide gel electrophoresis of low-molecular weight substances	18	(1)
Electrophoresis in restrictive gels	19	(22)
The Ferguson plot	19	(1)
Agarose gel electrophoresis	20	(1)
Polyacrylamide gel electrophoresis of nucleic acids	20	(12)
Polyacrylamide gel electrophoresis of proteins	32	(9)
Isotachophoresis	41	(4)
Migration with the same speed	41	(1)
``Ion train'' Separation	41	(1)
Zone sharpening effect	42	(1)
Concentration regulation effect	42	(3)
Isoelectric focusing	45	(14)
Principles	45	(2)
Gels for IEF	47	(1)
Temperature	48	(1)
Controlling the pH gradient	48	(1)
The kinds of pH gradients	48	(7)
Free carrier ampholytes	48	(4)
Immobilized pH gradients	52	(3)
Preparative isoelectric focusing	55	(1)
Titration curve analysis	56	(3)
Blotting	59	(12)
Principle	59	(1)
Transfer methods	59	(4)
Blotting membranes	63	(1)
Buffers for electrophoretic transfers	64	(2)
General staining	66	(1)
Blocking	66	(1)
Specific detection	67	(2)
Protein sequencing	69	(1)
Transfer problems	69	(2)
Interpretation of electropherograms	71	(10)
Introduction	71	(2)
Purity control	71	(1)
Quantification prerequisites	71	(2)
Image analysis	73	(8)
Hardware for image analysis	74	(1)
Software for image analysis	75	(6)
Proteome Analysis	81	(20)
General	81	(3)
Sample preparation	84	(1)
Two-dimensional electrophoresis	85	(3)
Detection techniques	88	(2)
Image analysis	90	(1)
Protein spot identification	90	(9)
Mass spectrometry methods	92	(5)
Peptide mass fingerprinting	97	(2)
Protein characterization	99	(1)
Bioinformatics	99	(1)
Functional proteomics	100	(1)
Instrumentation	101	(12)
Current and voltage conditions	101	(2)
Power supply	103	(1)
Separation chambers	103	(4)
Vertical apparatus	103	(1)
Horizontal apparatus	104	(3)
Staining apparatus for gels and blots	107	(1)
Automated electrophoresis	107	(2)
Instruments for 2-D electrophoresis	109	(1)
Isoelectric focusing apparatus	109	(1)
Multiple slab gel apparatus	110	(1)
Safety measures	110	(1)
Environmental aspects	111	(2)
Equipment for Part II	113	(228)
Instrumentation	113	(2)
Laboratory equipment	115	(1)
Consumables	116	(1)
Chemicals	117	(6)
Part II Methods
Page of dyes	123	(6)
Sample preparation	123	(1)
Stock solutions	123	(1)
Preparing the casting cassette	123	(3)
Casting the ultrathin-layer gels	126	(1)
Electrophoretic separation	126	(3)
Agarose and immuno electrophoresis	129	(12)
Sample preparation	129	(1)
Stock solutions	129	(1)
Preparing the gels	130	(4)
Electrophoresis	134	(3)
Protein detection	137	(4)
Titration curve analysis	141	(10)
Sample preparation	141	(1)
Stock solutions	141	(1)
Preparing the blank gels	142	(3)
Titration curve analysis	145	(2)
Coomassie and silver staining	147	(2)
Interpreting the curves	149	(2)
Native PAGE in amphoteric buffers	151	(12)
Sample preparation	152	(1)
Stock solutions	152	(3)
Preparing the empty gels	155	(2)
Electrophoresis	157	(3)
Coomassie and silver staining	160	(3)
Agarose IEF	163	(8)
Sample preparation	163	(1)
Preparing the agarose gel	164	(3)
Isoelectric focusing	167	(2)
Protein detection	169	(2)
PAGIEF in rehydrated gels	171	(12)
Sample preparation	171	(1)
Stock solutions	172	(1)
Preparing the blank gels	172	(3)
Isoelectric focusing	175	(2)
Coomassie and silver staining	177	(4)
Perspectives	181	(2)
Horizontal SDS-PAGE	183	(18)
Sample preparation	183	(4)
Stock solutions for the preparation of gels	187	(1)
Preparing the casting cassette	187	(2)
Gradient gel	189	(4)
Electrophoresis	193	(2)
Protein detection	195	(3)
Blotting	198	(1)
Perspectives	198	(3)
Vertical PAGE	201	(14)
Sample preparation	202	(1)
Stock solutions	202	(1)
Single gel casting	203	(4)
Multiple gel casting	207	(3)
Electrophoresis	210	(1)
SDS electrophoresis of small peptides	211	(2)
Two-dimensional electrophoresis	213	(1)
DNA electrophoresis	213	(1)
Long shelflife gels	214	(1)
Protein detection	214	(1)
Semi-dry blotting of proteins	215	(8)
Transfer buffers	216	(1)
Technical procedure	217	(4)
Staining of blotting membranes	221	(2)
IEF in immobilized pH gradients	223	(16)
Sample preparation	224	(1)
Stock solutions	224	(1)
Immobiline recipes	225	(3)
Preparing the casting cassette	228	(1)
Preparing the pH gradient gels	229	(5)
Isoelectric focusing	234	(2)
Coomassie and silver staining	236	(2)
Strategies for IPG focusing	238	(1)
High-resolution 2D electrophoresis	239	(22)
Sample preparation	240	(3)
Stock solutions	243	(1)
Preparing the gels	244	(4)
Separation conditions	248	(8)
Staining procedures	256	(5)
PAGE of double stranded DNA	261	(12)
Stock solutions	262	(1)
Preparing the gels	263	(3)
Sample preparation	266	(1)
Electrophoresis	267	(4)
Silver staining	271	(2)
Native PAGE of single stranded DNA	273	(6)
Sample treatment	275	(1)
Gell properties	276	(1)
Buffers and additives	276	(1)
Conditions for electrophoresis	277	(1)
Strategy for SSCP analysis	278	(1)
Denaturing gradient gel electrophoresis	279	(8)
Sample preparation	280	(1)
Rehydration solutions	280	(1)
Preparing the rehydration cassette	280	(2)
Rehydration	282	(2)
Electrophoresis	284	(2)
Silver staining	286	(1)
Denaturing PAGE of DNA	287	(54)
Sample preparation	288	(1)
Solutions	288	(1)
Rehydration	289	(1)
Electrophoresis	289	(3)
Silver staining	292	(1)
Appendix
A Trouble-shooting guide	293	(40)
A1 Isoelectric focusing	293	(1)
A1.1 PAGIEF with carrier ampholytes	293	(8)
A1.2 Agarose IEF with carrier ampholytes	301	(3)
A1.3 Immobilized pH gradients	304	(6)
A2 SDS electrophoresis	310	(8)
A3 Vertical PAGE	318	(2)
A4 Semi-dry blotting	320	(6)
A5 2-D electrophoresis	326	(4)
A6 DNA electrophoresis	330	(3)
B References	333	(8)
Index	341