Electrophoresis in Practice

by Westermeier, Reiner

• ISBN13: 9783527311811
• ISBN10: 3527311815
• Edition: 4th
• Format: Hardcover
• Copyright: 2005-03-04
• Publisher: Wiley-Blackwell

Summary

This laboratory guide for successful electrophoretic separations is divided into two parts to provide readers with a thorough presentation of the fundamentals followed by a detailed description of the most common methods currently in use.This fourth edition retains the successful concept of its predecessors, yet features a brand-new layout, and is further enhanced by a section on difference gel electrophoresis, while the chapter on proteome analysis is practically all new and considerably extended, plus there are now around 10 % new literature references.

Author Biography

Born in 1951, Reiner Westermeier gained his diploma in engineering in 1976, and his doctorate in 1981 at the Technical University of Munich, Germany.
He remained at the TU engaged in the development of new electrophoresis systems and applications until 1983, a period that also involved several international collaborations and lecturing tours.
From 1984 to 1987 Dr. Westermeier was employed as an electrophoresis products and applications specialist at LKB Instrument GmbH thereafter until 1990 at Pharmacia Biotech. He founded ETC Elektrophorese-Technik in 1991 for the development of new electrophoresis methods, media, and equipment. From January 1996 to July 1997 he was manager of scientific support Europe at Pharmacia Biotech in Freiburg, Germany. He has been at Amersham Pharmacia Biotech since July 1997, and its Senior Scientist for Proteomics since January 2002.
Reiner Westermeier is the author of several publications and two books, both published by WILEY-VCH.

Table of Contents

Foreword	XI
Preface	XIII
Abbreviations	XVII
Part I Fundamentals	1	(132)
Introduction	3	(6)
1 Electrophoresis	9	(36)
1.0 General	9	(8)
1.1 Electrophoresis in non-restrictive gels	17	(4)
1.1.1 Agarose gel electrophoresis	17	(3)
1.1.2 Polyacrylamide gel electrophoresis of low-molecular weight substances	20	(1)
1.2 Electrophoresis in restrictive gels	21	(24)
1.2.1 The Ferguson plot	21	(1)
1.2.2 Agarose gel electrophoresis	22	(2)
1.2.3 Polyacrylamide gel electrophoresis of nucleic acids	24	(10)
1.2.4 Polyacrylamide gel electrophoresis of proteins	34	(11)
2 Isotachophoresis	45	(6)
2.1 Migration with the same speed	45	(2)
2.2 "Ion train" separation	47	(1)
2.3 Zone sharpening effect	47	(1)
2.4 Concentration regulation effect	47	(4)
3 Isoelectric focusing	51	(16)
3.1 Principles	51	(2)
3.2 Gels for IEF	53	(1)
3.3 Temperature	54	(1)
3.4 Controlling the pH gradient	54	(1)
3.5 The kinds of pH gradients	55	(7)
3.5.1 Free carrier ampholytes	55	(4)
3.5.2 Immobilized pH gradients	59	(3)
3.6 Protein detection in IEF gels	62	(1)
3.7 Preparative isoelectric focusing	62	(2)
3.8 Titration curve analysis	64	(3)
4 Blotting	67	(14)
4.1 Principle	67	(1)
4.2 Transfer methods	68	(4)
4.2.1 Diffusion blotting	68	(1)
4.2.2 Capillary blotting	68	(1)
4.2.3 Pressure blotting	68	(1)
4.2.4 Vacuum blotting	69	(1)
4.2.5 Electrophoretic blotting	70	(2)
4.3 Blotting membranes	72	(1)
4.4 Buffers for electrophoretic transfers	73	(2)
4.5 General staining	75	(1)
4.6 Blocking	75	(1)
4.7 Specific detection	76	(2)
4.8 Protein sequencing	78	(1)
4.9 Transfer problems	79	(2)
5 Interpretation of electropherograms	81	(10)
5.1 Introduction	81	(2)
5.1.1 Purity control	81	(1)
5.1.2 Quantification prerequisites	81	(2)
5.2 Image analysis	83	(8)
5.2.1 Hardware for image analysis	84	(3)
5.2.2 Software for image analysis	87	(4)
6 Proteome Analysis	91	(30)
6.1 General	91	(5)
6.2 Sample preparation	96	(4)
6.3 Two-dimensional electrophoresis	100	(2)
6.4 Detection techniques	102	(5)
6.5 Image analysis	107	(2)
6.6 Protein spot identification	109	(9)
6.6.1 Mass spectrometry methods	111	(5)
6.6.2 Peptide mass fingerprinting	116	(2)
6.6.3 Protein characterization	118	(1)
6.7 Bioinformatics	118	(1)
6.8 Functional proteomics	119	(2)
7 Instrumentation	121	(12)
7.1 Current and voltage conditions	121	(2)
7.2 Power supply	123	(1)
7.3 Separation chambers	124	(4)
7.3.1 Vertical apparatus	124	(1)
7.3.2 Horizontal apparatus	125	(3)
7.4 Staining apparatus for gels and blots	128	(1)
7.5 Automated electrophoresis	128	(2)
7.6 Instruments for 2-D electrophoresis	130	(1)
7.6.1 Isoelectric focusing apparatus	130	(1)
7.6.2 Multiple slab gel apparatus	131	(1)
7.7 Safety measures	131	(1)
7.8 Environmental aspects	132	(1)
Part II Equipment and Methods	133	(206)
Equipment for Part II	135	(10)
Instrumentation	136	(2)
Special laboratory equipment	138	(1)
Consumables	139	(1)
Chemicals	140	(5)
Method 1: PAGE of dyes	145	(6)
1 Sample preparation	145	(1)
2 Stock solutions	145	(1)
3 Preparing the casting cassette	146	(2)
4 Casting ultrathin-layer gels	148	(1)
5 Electrophoretic separation	149	(2)
Method 2: Agarose and immuno electrophoresis	151	(12)
1 Sample preparation	151	(1)
2 Stock solutions	152	(1)
3 Preparing the gels	152	(5)
4 Electrophoresis	157	(2)
5 Protein detection	159	(4)
Method 3: Titration curve analysis	163	(12)
1 Sample preparation	163	(1)
2 Stock solutions	164	(1)
3 Preparing the blank gels	164	(3)
4 Titration curve analysis	167	(3)
5 Coomassie and silver staining	170	(2)
6 Interpreting the curves	172	(3)
Method 4: Native PAGE in amphoteric buffers	175	(14)
1 Sample preparation	176	(1)
2 Stock solutions	177	(1)
3 Preparing the empty gels	178	(4)
4 Electrophoresis	182	(3)
5 Coomassie and silver staining	185	(4)
Method 5: Agarose IEF	189	(8)
1 Sample preparation	189	(1)
2 Preparing the agarose gel	190	(3)
3 Isoelectric focusing	193	(2)
4 Protein detection	195	(2)
Method 6: PAGIEF in rehydrated gels	197	(14)
1 Sample preparation	198	(1)
2 Stock solutions	198	(1)
3 Preparing the blank gels	199	(2)
4 Isoelectric focusing	201	(3)
5 Coomassie and silver staining	204	(4)
6 Perspectives	208	(3)
Method 7: Horizontal SDS-PAGE	211	(20)
1 Sample preparation	211	(4)
2 Stock solutions for gel preparation	215	(1)
3 Preparing the casting cassette	216	(2)
4 Gradient gel	218	(3)
5 Electrophoresis	221	(3)
6 Protein detection	224	(3)
7 Blotting	227	(1)
8 Perspectives	227	(4)
Method 8: Vertical PAGE	231	(16)
1 Sample preparation	232	(1)
2 Stock solutions	233	(1)
3 Single gel casting	233	(4)
4 Multiple gel casting	237	(4)
5 Electrophoresis	241	(1)
6 SDS electrophoresis of small peptides	242	(2)
7 Two-dimensional electrophoresis	244	(1)
8 DNA electrophoresis	244	(1)
9 Long shelflife gels	245	(1)
10 Detection of bands	245	(2)
Method 9: Semi-dry blotting of proteins	247	(10)
1 Transfer buffers	248	(1)
2 Technical procedure	249	(4)
3 Staining of blotting membranes	253	(4)
Method 10: IEF in immobilized pH gradients	257	(18)
1 Sample preparation	258	(1)
2 Stock solutions	258	(1)
3 Immobiline recipes	259	(3)
4 Preparing the casting cassette	262	(2)
5 Preparing the pH gradient gels	264	(5)
6 Isoelectric focusing	269	(2)
7 Staining	271	(2)
8 Strategies for IPG focusing	273	(2)
Method 11: High-resolution 2-D electrophoresis	275	(24)
1 Sample preparation	276	(3)
2 Stock solutions	279	(1)
3 Preparing the gels	280	(5)
4 Separation conditions	285	(8)
5 Staining procedures	293	(6)
Method 12: PAGE of double stranded DNA	299	(14)
1 Stock solutions	300	(1)
2 Preparing the gels	301	(4)
3 Sample preparation	305	(1)
4 Electrophoresis	305	(5)
5 Silver staining	310	(3)
Method 13: Native PAGE of single stranded DNA	313	(8)
1 Sample treatment	315	(1)
2 Gel properties	316	(1)
3 Buffers and additives	317	(1)
4 Conditions for electrophoresis	318	(1)
5 Strategy for SSCP analysis	318	(3)
Method 14: Denaturing gradient gel electrophoresis	321	(10)
1 Sample preparation	322	(1)
2 Rehydration solutions	322	(1)
3 Preparing the rehydration cassette	323	(2)
4 Rehydration	325	(2)
5 Electrophoresis	327	(2)
6 Silver staining	329	(2)
Method 15: Denaturing PAGE of DNA	331	(8)
1 Sample preparation	332	(1)
2 Solutions	333	(1)
3 Rehydration	333	(1)
4 Electrophoresis	334	(3)
5 Silver staining	337	(2)
Appendix Trouble-shooting	339	(50)
A1 Isoelectric focusing	339	(20)
A1.1 PAGIEF with carrier ampholytes	339	(9)
A1.2 Agarose IEF with carrier ampholytes	348	(4)
A1.3 Immobilized pH gradients	352	(7)
A2 SDS electrophoresis	359	(10)
A3 Vertical PAGE	369	(2)
A4 Semidry blotting	371	(8)
A5 2-D electrophoresis	379	(6)
A6 DNA electrophoresis	385	(4)
References	389	(10)
Index	399