Table of Contents

Cover

Title Page

Foreword

Abbreviations, Symbols, Units

Preface

German Version
English Version, Fifth Edition

Part I: Fundamentals

Chapter 1: Electrophoresis
1. 1.1 General
2. 1.2 Electrophoresis in Nonrestrictive Gels
3. 1.3 Electrophoresis in Restrictive Gels
4. References
Chapter 2: Isotachophoresis
1. 2.1 Migration with the Same Speed
2. 2.2 “Ion Train” Separation
3. 2.3 Zone Sharpening Effect
4. 2.4 Concentration Regulation Effect
5. 2.5 Quantitative Analysis
6. References
Chapter 3: Isoelectric Focusing
1. 3.1 Principles
2. 3.2 Gels for IEF
3. 3.3 Temperature
4. 3.4 Controlling the pH Gradient
5. 3.5 Kinds of pH Gradients
6. 3.6 Protein Detection in IEF Gels
7. 3.7 Preparative Isoelectric Focusing
8. 3.8 Titration Curve Analysis
9. References
Chapter 4: High-Resolution Two-Dimensional Electrophoresis
1. 4.1 IEF in Immobilized pH Gradient Strips
2. 4.2 SDS-PAGE
3. 4.3 Proteomics
4. References
Chapter 5: Protein Sample Preparation
1. 5.1 Protein Quantification Methods
2. 5.2 Preparation of Native Samples
3. 5.3 Samples for SDS Electrophoresis
4. 5.4 Samples for High-Resolution 2D PAGE
5. References
Chapter 6: Protein Detection
1. 6.1 Fixation
2. 6.2 Poststaining Methods
3. 6.3 Prelabeling
4. 6.4 Difference Gel Electrophoresis (DIGE)
5. 6.5 Imaging, Image Analysis, Spot Picking
6. References
Chapter 7: Blotting
1. 7.1 Transfer Methods
2. 7.2 Blotting Membranes
3. 7.3 Buffers for Electrophoretic Transfers
4. 7.4 General Staining
5. 7.5 Blocking
6. 7.6 Specific Detection
7. 7.7 Protein Sequencing
8. 7.8 Transfer Issues
9. 7.9 Electro-Elution of Proteins from Gels
10. References

Part II: Equipment and Methods

Chapter 8: Special Laboratory Equipment
Chapter 9: Consumables
Chapter 10: Chemicals
1. 10.1 Reagents
Method 1: PAGE of Dyes
1. M1.1 Sample Preparation
2. M1.2 Stock Solutions
3. M1.3 Preparing the Casting Cassette
4. M1.4 Casting Ultra-Thin-Layer Gels
5. M1.5 Electrophoretic Separation
Method 2: Agarose and Immunoelectrophoresis
1. M2.1 Sample Preparation
2. M2.2 Stock Solutions
3. M2.3 Preparing the Gels
4. M2.4 Electrophoresis
5. M2.5 Protein Detection
6. References
Method 3: Titration Curve Analysis
1. M3.1 Sample Preparation
2. M3.2 Stock Solutions
3. M3.3 Preparing the Blank Gels
4. M3.4 Titration Curve Analysis
5. M3.5 Coomassie and Silver Staining
6. M3.6 Interpreting the Curves
7. References
Method 4: Native PAGE in Amphoteric-Buffers
1. M4.1 Sample Preparation
2. M4.2 Stock Solutions
3. M4.3 Preparing the Empty Gels
4. M4.4 Electrophoresis
5. M4.5 Coomassie and Silver Staining
6. References
Method 5: Agarose IEF
1. M5.1 Sample Preparation
2. M5.2 Preparing the Agarose Gel
3. M5.3 Isoelectric Focusing
4. M5.4 Protein Detection
5. References
Method 6: PAGIEF in Rehydrated Gels
1. M6.1 Sample Preparation
2. M6.2 Stock Solutions
3. M6.3 Preparing the Blank Gels
4. M6.4 Isoelectric Focusing
5. M6.5 Coomassie and Silver Staining
6. M6.6 Perspectives
7. References
Method 7: Horizontal SDS-PAGE
1. M7.1 Sample Preparation
2. M7.2 Prelabeling with Fluorescent Dye
3. M7.3 Stock Solutions for Gel Preparation
4. M7.4 Preparing the Casting Cassette
5. M7.5 Gradient Gel
6. M7.6 Electrophoresis
7. M7.7 Protein Detection
8. M7.8 Blotting
9. M7.9 Perspectives
10. References
Method 8: Vertical PAGE
1. 8.1 Sample Preparation and Prelabeling
2. 8.2 Stock Solutions for SDS- PAGE
3. 8.3 Single Gel Casting
4. 8.4 Multiple Gel Casting
5. 8.5 Electrophoresis
6. 8.6 SDS Electrophoresis of Small Peptides
7. 8.7 Blue Native PAGE
8. 8.8 Two-Dimensional Electrophoresis
9. 8.9 DNA Electrophoresis
10. 8.10 Long-Shelf-Life Gels
11. 8.11 Protein Detection
12. 8.12 Preparing Glass Plates with Bind-Silane
13. References
Method 9: Semidry Blotting of Proteins
1. M9.1 Transfer Buffers
2. M9.2 Technical Procedure
3. M9.3 Staining of Blotting Membranes
4. References
Method 10: IEF in Immobilized pH Gradients
1. M10.1 Sample Preparation
2. M10.2 Stock Solutions
3. M10.3 Immobiline Recipes
4. M10.4 Preparing the Casting Cassette
5. M10.5 Preparing the pH Gradient Gels
6. M10.6 Isoelectric Focusing
7. M10.7 Staining
8. M10.8 Strategies for IPG Focusing
9. References
Method 11: High-Resolution 2D Electrophoresis
1. M11.1 Sample Preparation
2. M11.2 Prelabeling of Proteins with Fluorescent Dyes
3. M11.3 Stock Solutions for Gel Preparation
4. M11.4 Preparing the Gels
5. M11.5 Separation Conditions
6. M11.6 Staining Procedures
7. References
Method 12: PAGE of DNA Fragments
1. M12.1 Stock Solutions
2. M12.2 Preparing the Gels
3. M12.3 Sample Preparation
4. M12.4 Electrophoresis
5. M12.5 Silver Staining

Appendix: Troubleshooting

A1.1 Frequent Mistakes
A1.2 Isoelectric Focusing
A1.3 SDS Electrophoresis
A1.4 Two-Dimensional Electrophoresis
A1.5 Semi-Dry Blotting
A1.6 DNA Electrophoresis
1. Index

End User License Agreement

List of Illustrations

Chapter 1: Electrophoresis

Figure 1.1 Moving boundary electrophoresis in a U-shaped tube according to Tiselius. Measurements of the electrophoretic mobilities are done with Schlieren optics on both ends.
Figure 1.2 Schematic drawing of a continuous free-flow electrophoresis system.
Figure 1.3 Schematic representation of the set-up of capillary electrophoresis.
Figure 1.4 Microchip electrophoresis. For details see text.
Figure 1.5 Chemical structure of agarose and structure of the polysaccharide polymers after gel formation.
Figure 1.6 Polymerization reaction of acrylamide and methylenebisacrylamide.
Figure 1.7 Gel geometries for electrophoretic separations.
Figure 1.8 Schematic diagram of the relationships between the separation medium and current, voltage, and power conditions during electrophoresis.
Figure 1.9 Course of electric parameters during disc electrophoresis and isoelectric focusing.
Figure 1.10 Different designs of vertical chamber systems: (a) Simple noncooled setup with upper and lower buffer reservoir. (b) System with large “upper” buffer tank acting also as a heat sink, optionally with cooling via tubing. (c) System with large lower buffer tank acting also as a heat sink optionally with cooling via tubing. (d) Cassette design for set-ups (a) and (b) with a notched glass plate. (e) Plastic cassette containing readymade gel. (f) Casting stand with cassette for set-up (c) with two glass plates with even upper edges.
Figure 1.11 Submarine chamber. (a) Casting tray. (b) Agarose chamber during electrophoresis.
Figure 1.12 System for pulsed-field DNA gel electrophoresis (PFG). (a) PFG submarine chamber with cooling coil and buffer circulation pump (not visible). (b) Programmable pulse controlling device. (c) Hexagonal electrode set-up for linear sample lanes.
Figure 1.13 Different designs of horizontal flatbed electrophoresis systems. (a) Separation chamber with cooling plate and lateral buffer tanks. (b) Separation cabinet chamber without buffer tanks and a drawer-cooling plate. (c) Gel casting cassette for thin, homogeneous and gradient gels. (d) Set-up of a horizontal gel on film backing with electrode wicks containing concentrated buffer.
Figure 1.14 Agarose gel electrophoresis of lactate dehydrogenase isoenzymes. Specific staining was performed with zymogram technique.
Figure 1.15 Three principles of immunoelectrophoresis, see text for details.
Figure 1.16 Affinity electrophoresis of human alkaline phosphatase isoenzymes from liver and bones. The gel contains the lectin wheat germ agglutinin, which specifically interacts with the bone fraction. The retarded zone can be recognized as a characteristic band close to the application point. Alkaline phosphatase zymogram detection.
Figure 1.17 Ferguson plots: plots of the electrophoretic migrations of proteins versus gel concentrations. (a) Lactate dehydrogenase isoenzymes and (b) different proteins. See text for further details.
Figure 1.18 Set-up for the “submarine” technique for the separation of nucleic acids.
Figure 1.19 Field lines and separation results for two types of PFG electrophoresis: (left) orthogonal doubly inhomogeneous fields, and (right) homogeneous fields for hexagonally arranged point electrodes.
Figure 1.20 Instrumentation for automated DNA sequencing with a four-track system. On the computer screen is seen a typical trace after treatment of the crude data by a computer.
Figure 1.21 RAPD electrophoresis of fungi varieties in a horizontal polyacrylamide gel. Silver staining. By kind permission of Birgit Jäger and Dr Hans-Volker Tichy, TÜV Südwest GmbH – Biological Safety Division, Freiburg im Breisgau.
Figure 1.22 Schematic representation of typical results of a perpendicular and a parallel DGGE.
Figure 1.23 Schematic diagram of the principles of disc electrophoresis according to Ornstein (1964). The buffer system shown is also employed for discontinuous SDS electrophoresis.
Figure 1.24 Casting of gradient gels with a gradient maker. The stirrer bar is rotated with a magnetic stirrer (not shown). (a) Linear gradient. (b) Exponential gradient.
Figure 1.25 Separation of proteins in a horizontal SDS polyacrylamide gel T = 12.5% (cathode on top). Stained with Coomassie Brilliant Blue R 250. Samples: human serum, leguminosae seed extracts and different marker protein mixtures.
Figure 1.26 Semilogarithmic representation of a molecular weight curve. The molecular weights of the marker proteins are represented as a function of their migration. (SDS linear pore gradient gel according to Figure 1.21.)
Figure 1.27 Principle of the buffer systems of readymade gels for discontinuous SDS electrophoresis. Horizontal gels with Tris–tricine buffer strips.
Figure 1.28 Principle of two-dimensional electrophoresis with blue native PAGE in the first and SDS-PAGE in the second dimension.
Figure 1.29 Principle of two-dimensional electrophoresis with a cationic detergent 16-BAC acidic PAGE in the first and SDS-PAGE in the second dimension.
Figure 1.30 Principle of the original high-resolution 2-D electrophoresis according to O'Farrell (1975).

Chapter 2: Isotachophoresis

Figure 2.1 The four facts of isotachophoresis. (a) In a discontinuous buffer system the ions are forced to migrate with the same speed. (b) The ions are separated, but each zone travels in immediately after the other one. (c) Because of the slowing down and acceleration of ions in the different field strength areas, zones are sharpened. (d) The concentration regulating effect is the basis of quantification in isotachophoresis, see text for further explanations.
Figure 2.2 Isotachophoresis of penicillin. Simultaneous detection of the zones with a thermocouple detector and an UV detector. From the application laboratory of Pharmacia LKB, Sweden.

Chapter 3: Isoelectric Focusing

Figure 3.1 Protein molecule and the dependence of the net charge on the pH value. A protein with this net charge has two positive charges at pH 6 and one negative charge at pH 9.
Figure 3.2 Several ways of sample application on horizontal gel surface. In this example, electrode strips are applied: (a) droplets, (b) applicator pieces, (c) applicator strip, and (d) Raschig rings.
Figure 3.3 Schematic representation of the formation of a carrier ampholyte pH gradient in the electric field.
Figure 3.4 Isoelectric focusing in a washed and rehydrated polyacrylamide gel. Press sap of potatoes of different cultivars. Coomassie Brilliant Blue staining (anode on top).
Figure 3.5 Diagram of a polyacrylamide network with copolymerized Immobilines.
Figure 3.6 IEF in immobilized pH gradient (pH 4.0–5.0). Isoforms of α1-antitrypsin (protease inhibitors) in human serum. By kind permission of Prof Dr Pollack and Ms Pack, Institut für Rechtsmedizin der Universität Freiburg im Breisgau (anode at the top).
Figure 3.7 Purification of a protein (a) between two isoelectric membranes. All contaminating charged substances and proteins with pIs lower than pH 5.4 (b) or higher than pH 5.6 (c, d) migrate out of the central chamber, which is enclosed by the two membranes.
Figure 3.8 Principle of Off-gel isoelectric focusing. The sample components are separated according to their isoelectric points and enriched on the surface of an IPG strip in the liquid phase. The diluted samples are pipetted into the compartments of a fractionating frame. In the electric field, the charged proteins or peptides migrate through the gel layer of the IPG strip until they reach the pH values corresponding to their pIs.
Figure 3.9 Titration curves. (a) Application of the sample in the sample trench after the pH gradient has been established. (b) Titration curves.
Figure 3.10 Titration curves of a pI marker protein mixture (pH 3–10). Cathode is at the top.

Chapter 4: High-Resolution Two-Dimensional Electrophoresis

Figure 4.1 Schematic drawing of 2D electrophoresis with IPG strips. The second dimension can be run in a flatbed or a vertical tank system.
Figure 4.2 Two-dimensional electrophoresis of mouse liver proteins. First dimension: isoelectric focusing in an immobilized pH gradient pH 3–12 in a 24 cm long gel strip. Second dimension: SDS-PAGE in a 13% gel. Silver stained. (With kind permission of Professor A. Görg, Technische Universität München-Weihenstephan.)
Figure 4.3 Result of 2D PAGE of a sample with too high a salt content (after washing cells with phosphate buffered saline, PBS). First dimension in an IPG gradient 3–10, second dimension SDS-PAGE, Coomassie Blue staining. The high amounts of salt anions and cations cause the stop of ion migration due to the very high conductivity differences. E = electric field strength, U = voltage, d = protein migration distance.
Figure 4.4 Steps for rehydration of IPG strips in individual grooves of a reswelling tray.
Figure 4.5 Schematic drawing of different possibilities of sample application on an IPG strip: (a) Cup-loading for up to 250 µl sample solution. (b) Paper bridge loading for up to 500 µl sample solution. For (a) and (b), anodal application is shown as an example; for some samples and gradients, sample application at the cathode end might be preferable. (c) Rehydration loading and running in a manifold. (d) Rehydration loading in an individual strip holder for active rehydration and automated running.
Figure 4.6 Evolution of instruments for running IPG strips. (a) Accessory kit for the Multiphor chamber. (b) IPGphor with single strip holders for rehydration loading ((a) and (b) available from GE Healthcare). (c) IEF 100 (by Hoefer Inc.) for six strips with individual current recording. (d) Protean i12 IEF system (by Biorad) with 12 individually powered channels. Panels (b)–(d) are equipped with inbuilt Peltier cooling and programmable power supply.
Figure 4.7 Different ways to run the second dimension: (a) Vertical single buffer tank for running the separation sideways. (b) Vertical dual tank system for running the separation from top down. (c) Horizontal system “HPE™ Tower” with drawer-cooling plates using buffer wicks instead of buffer tanks.
Figure 4.8 Preparing a film-backed flatbed gel for 2D PAGE and loading the IPG strip. (a) Soaking the electrode wicks with the electrode buffer concentrates. (b) Application of the cooling contact fluid on the cooling plate. (c) Distributing the cooling contact fluid evenly and placing the gel on the cooling plate. (d) Inserting the equilibrated IPG strip into the groove.
Figure 4.9 (a) Glass cassette for multiple large-format gel casters and separation chambers. Spacers are fixed to the glass surface, and the glass plates are connected with a hinge. (b) Casting multiple homogeneous SDS gels by pouring the monomer solution directly into the upper filling channel. The lower filling port is used for casting multiple gradient gels.
Figure 4.10 Application of IPG strip and SDS size markers on the SDS gel and embedding with hot agarose.

Chapter 5: Protein Sample Preparation

Figure 5.1 Native structure of proteins.
Figure 5.2 Proteins treated with SDS without any reducing agent.
Figure 5.3 SDS-treated and reduced proteins.
Figure 5.4 Reduced and alkylated proteins treated with SDS.

Chapter 6: Protein Detection

Figure 6.1 Diagram of an excitation and an emission curve of a fluorophore.
Figure 6.2 SDS electrophoresis in SERVAGel™ PRiME TG 4–20. Lanes 1–6: SERVA-Protein test mix 6: 5 µg, 1 µg, 0.2 µg, 40 ng, 8 ng, and 1.6 ng. Lanes 7–12: Escherichia coli lysate with protein contents of 25 µg, 5 µg, 1 µg, 0.5 µg, 250 ng, and 125 ng. Proteins were detected with (a) staining with Coomassie Brilliant Blue R 250, (b) silver staining, (c) staining with ServaPurple, and (d) prelabeling with Serva LightningRed. (c) and (d) scanned with 532 nm excitation and 630 nm emission wavelength using a narrow band-pass filter. (From Griebel et al. (2013a). With permission of Springer Science + Business Media.)
Figure 6.3 Workflow of protein detection methods. (Modified from Griebel et al. (2013b). With permission of Springer Science + Business Media.)
Figure 6.4 Difference gel electrophoresis (DIGE): (a) Schematic representation of the concept of labeling two samples and a mixture of the samples, a pooled standard, with three different fluorescent dyes, and running them together in 2D electrophoresis. (b) False-color representation of the Cy3 and Cy5 image channels after scanning and overlaying of the images. Yellow spots: no changes; red spots: up-regulated; green spots: down-regulated proteins. “Healthy” and “diseased” can be replaced by “control” and “treated” or by “wild type” and “mutant.”
Figure 6.5 Comparative fluorescence gel electrophoresis in a horizontal gel. 50 µg E. coli: red; 0.72 µg reference grid mixture per well: green). (From Hanneken and König, 2014, with kind permission of M. Hannecken and S. König, IZKF Universität Münster.)
Figure 6.6 Color intensity curve of carbonic anhydrase, separated with SDS-PAGE and stained with colloidal Coomassie Brilliant Blue G-250.
Figure 6.7 Diagram of the protein amount of individual markers per integrated peak area. TI: trypsin inhibitor from soybean. Staining with Coomassie Brilliant Blue G-250.
Figure 6.8 Scanners for electrophoresis gels. (a) Desktop A4 scanner for visible dyes in transmission and reflection mode, Bio5000 (SERVA and Microtek). (b) Multipurpose scanner Typhoon (GE Healthcare) with confocal optics for fluorescence detection in gels at different wavelengths and storage phosphor plate imaging.
Figure 6.9 CCD camera system with new diffuse lighting technology for visible and UV dyes, chemiluminescence and fluorescence imaging at different wavelengths. WiFi enabled SERVA Blue Imager (SERVA).
Figure 6.10 Computer screen showing the detected lanes of an SDS gel and the densitogram of a selected lane with annotated molecular weights.
Figure 6.11 Computer screen of 2D electrophoresis image analysis software. In the background are 2D images with automatically detected spots. In the foreground are volume table, histograms and 3D representation of selected spots.
Figure 6.12 ScreenPicker for semiautomated picking of spots from fluorescent and visible stained gels. The image is projected with a flat screen below the real gel lying on a glass platen. Spot plugs are excised with a manual picker and transferred to the well in the microtiter plate at the indicated position. RM: reference marker.

Chapter 7: Blotting

Figure 7.1 Diagram of the most important steps during blotting from electrophoresis gels.
Figure 7.2 Bidirectional transfer of proteins by diffusion blotting from a gel with large pores.
Figure 7.3 Capillary blotting; the transfer occurs overnight.
Figure 7.4 Pressure blotting of a film-backed gel.
Figure 7.5 Transfer of nucleic acids with vacuum blotting in 30–40 min.
Figure 7.6 Schematic diagram of a tank blotter.
Figure 7.7 Examples of semidry blotters: (a) Stand-alone blotter TE77XP (Hoefer) with weight. (b) Trans-Blot® Turbo™ Transfer System (BioRad) with two cassettes. (c) iBlot® (life technologies).
Figure 7.8 Diagram of a horizontal graphite blotter for semidry blotting. (Modified from Kyhse-Andersen (1984).
Figure 7.9 Instrument for complete and trouble-free separation of gels backed by films. (Available from Serva or Proteomics Consult.)
Figure 7.10 Diagram of fluorescent blotting.
Figure 7.11 Schematic diagram of enzymatic chemiluminescence detection plus biotin–streptavidin complexes, which afford the highest detection sensitivity.
Figure 7.12 Schematic representation of the “Simple Western” approach.
Figure 7.13 Approaches for electro-elution of proteins working without dialysis membranes. (a) The gel piece is placed into a conical inner elution tube, which is inserted into a 1.5-ml reaction cup, and closed with an electrode cap. (b) Electro-elution into a discontinuous conductivity gradient.

Method 1: PAGE of Dyes

Figure M1.1 Preparing the slot-former.
Figure M1.2 Applying the support film with a roller.
Figure M1.3 Assembling the gel cassette.
Figure M1.4 Casting an ultra-thin-layer polyacrylamide gel. After the calculated amount of polymerization solution has been added, remove the paper clips and push the clamps back into position.
Figure M1.5 Soaking the wicks in electrode buffer in the PaperPool. Roll out air bubbles.
Figure M1.6 Gel and electrode wicks for the separation of dyes. The samples are applied in the middle. Example of a horizontal chamber: Multiphor II. Dotted lines on the electrode wicks indicate the positions where the electrodes should be placed.
Figure M1.7 Ultra-thin-layer polyacrylamide gel electrophoresis of dyes. Anode is at the top.

Method 2: Agarose and Immunoelectrophoresis

Figure M2.1 Preparing the slot former.
Figure M2.2 Applying the support film with a roller.
Figure M2.3 Assembling the gel cassette.
Figure M2.4 Pouring the hot agarose solution into the prewarmed cassette.
Figure M2.5 Storing the agarose gel overnight in a humidity chamber.
Figure M2.10 Large immunoelectrophoresis gel with the Grabar–Williams technique.
Figure M2.6 Punching out the sample wells and cutting the troughs.
Figure M2.7 Punching out the sample wells for rocket immunoelectrophoresis.
Figure M2.8 Drying the surface of the gel with filter paper.
Figure M2.9 Agarose gel electrophoresis. Example of a horizontal chamber: Multiphor II.
Figure M2.11 Pressing the agarose gel.

Method 3: Titration Curve Analysis

Figure M3.1 Cutting the narrow adhesive tape to form the sample trenches in the gel.
Figure M3.2 Applying the support film with a roller.
Figure M3.3 Assembling the gel cassette.
Figure M3.5 Rehydration of a titration curve gel in the GelPool.
Figure M3.6 Placing the titration gel on the cooling plate for the formation of the pH gradient. Example for a horizontal chamber: Blue Horizon.
Figure M3.7 Schematic representation of titration curves.

Method 4: Native PAGE in Amphoteric-Buffers

Figure M4.1 Preparing the slot former.
Figure M4.2 Applying the support film with a roller.
Figure M4.3 Assembling the gel cassette.
Figure M4.4 Pouring the polymerization solution.
Figure M4.5 Rehydration of a gel. (a) Placing the dry gel into the GelPool. (b) Lifting the gel for an even spreading of the liquid. (c) Rehydration on a rocking platform (not always necessary). (d) Removing the excess buffer from the gel surface with filter paper.
Figure M4.6 Electrophoresis of cationic dyes in 0.5 mol/L HEPES, method 4a.
Figure M4.7 Soaking the wicks in electrode buffer in the PaperPool. Roll out air bubbles.
Figure M4.8 Cationic native electrophoresis: sample application at the anode. Dotted lines on the electrode wicks indicate the positions where the electrodes should be placed. Example of a horizontal chamber: Blue Horizon.
Figure M4.9 Cationic native electrophoresis of marker proteins and meat extracts in 0.6 mol l⁻¹ HEPES. Staining with Coomassie Brilliant Blue R-250 (cathode on top).

Method 5: Agarose IEF

Figure M5.1 Applying the support film with a roller.
Figure M5.2 Assembling the gel cassette.
Figure M5.3 Pouring the hot agarose solution into the preheated cassette.
Figure M5.4 Storing the agarose gel overnight in a humidity chamber.
Figure M5.5 Drying the surface of the gel with filter paper.
Figure M5.6 Agarose IEF with electrode strips and sample application strip. Example for a horizontal chamber: Blue Horizon.
Figure M5.7 Pressing the agarose gel.

Method 6: PAGIEF in Rehydrated Gels

Figure M6.1 Applying the support film with a roller.
Figure M6.2 Assembling the gel cassette.
Figure M6.4 Rehydration of an IEF gel. (a) Placing the dry gel into the GelPool. (b) Lifting the gel for an even spreading of the liquid. (c) Rehydration on a rocking platform (not always necessary). (d) Removing the excess buffer from the gel surface with filter paper.
Figure M6.5 For washed IEF gels, the focusing electrodes are placed directly on the edges of the gel. Example for a horizontal chamber: Blue Horizon.
Figure M6.6 Step trial test to determine the best application point.

Method 7: Horizontal SDS-PAGE

Figure M7.1 SDS PAGE of fluorescent-labeled urinary proteins in a horizontal gel. (a) Fluorescent image taken within 4 s with the Serva BlueImager using blue LEDs for excitation and an emission filter with 595 nm. (b) Silver staining of the same gel. (From Serva development laboratory with kind permission.)
Figure M7.2 Preparing the slot former.
Figure M7.3 Applying the support film with a roller.
Figure M7.4 Assembling the gel cassette.
Figure M7.5 Set-up of gel and electrode wicks for semi-dry SDS electrophoresis. The dotted lines indicate the position for the electrode contacts. Example for a horizontal chamber: Blue Horizon.
Figure M7.6 Set-up for hot staining.
Figure M7.7 Pouring the linear gradient.
Figure M7.8 Pouring the very dense monomer solution for the plateau.
Figure M7.9 Soaking the wicks in electrode buffer in the PaperPool. Smooth out air bubbles with a roller.
Figure M7.10 (a) SDS electrophoresis of legume seed extracts and molecular weight standards in a rehydrated discontinuous gel with a 5%T stacking zone and a 10%T resolving zone. (b) Separation of peptide and low molecular weight markers in a rehydrated 15%T gel containing 30% (v/v) monoethylene glycol and the buffer described in the text. Staining is with Coomassie Brilliant Blue R-350.
Figure M7.11 Selective equilibration of the stacking gel zone in the appropriate buffer for disc electrophoresis.

Method 8: Vertical PAGE

Figure M8.1 Vertical SDS electrophoresis of legume seed extracts and markers in a minigel, modified procedure. Silver staining is used.
Figure M8.2 Gel cassette for a vertical gel.
Figure M8.3 Preparation of the gel casting stand for one- or two-gel cassettes.
Figure M8.4 Casting a gradient gel.
Figure M8.5 Assembling the gel cassettes into a stand for multiple gel casting.
Figure M8.6 Casting multiple discontinuous gels.
Figure M8.7 Casting multiple-gradient gels.
Figure M8.10 Casting a porosity gradient for a blue native-PAGE gel from the bottom.
Figure M8.11 Blue native-PAGE/SDS-PAGE electrophoresis of mitochondrial complexes of Arabidopsis thaliana (from Eubel, Jänsch and Braun, 2003). The complexes were solubilized with digitonin and are annotated with Roman numerals. I + III is a super complex composed of complexes I and III. Coomassie Brilliant Blue poststaining of the SDS gel.
Figure M8.12 Drying of a polyacrylamide gel between two sheets of cellophane using two plastic frames.

Method 9: Semidry Blotting of Proteins

Figure M9.1 Installing the graphite anode in place of the cooling plate. Example of a horizontal chamber: Multiphor with NovaBlot blotting unit.
Figure M9.2 Mask.
Figure M9.3 Assembling the blotting sandwich on the graphite anode plate.
Figure M9.4 Removing the support film and transferring the support film–gel–blotting membrane sandwich to the stack of anode paper.
Figure M9.5 Rolling out the air bubbles.
Figure M9.6 (left) Blot of an SDS electrophoretic separation of legume seed extracts on nitrocellulose; Indian ink stain. (right) Coomassie-stained gel containing the same protein pattern in the same concentration.

Method 10: IEF in Immobilized pH Gradients

Figure M10.1 Graphic interpolation of a custom-made immobilized pH gradient pH 5.3–5.6.
Figure M10.2 Applying the support film with a roller.
Figure M10.3 Assembling the gel cassette.
Figure M10.4 Pouring the linear pH gradient.
Figure M10.5 Rehydration of an IPG gel (a) in a vertical cassette, or, alternatively, (b) in the rehydration tray GelPool.
Figure M10.6 Rehydrating in an additive gradient using the reswelling cassette.
Figure M10.7 Placing the Immobiline gel on the cooling plate of the separation chamber.
Figure M10.8 Possibilities for sample application for IPG. (1) Droplets. (2) Pieces of cellulose. (3) Sample application mask. (4) Cut-off silicone tubing.
Figure M10.9

Method 11: High-Resolution 2D Electrophoresis

Figure M11.1 Two 2D separations of the same sample: (left) Crude sample applied by rehydration loading. (right) Sample applied after a clean-up with precipitation. Silver staining.
Figure M11.2 Casting an IPG gel for long separation distance.
Figure M11.3 Precise cutting of the dry IPG strips with a rotary paper cutter.
Figure M11.4 Rehydration tray for reswelling the IPG strips in grooves, either in the rehydration solution (for cup loading) or in the sample solution (rehydration loading).
Figure M11.5 IEF in individual strips directly on the cooling plate. The open gel surfaces are covered with parafilm. Example for a horizontal chamber: Blue Horizon.
Figure M11.6 IEF in individual IPG strips in the IPG strip kit, which is an accessory of the Multiphor chamber.
Figure M11.7 Rehydration loading in individual ceramic trays. (a) Pipetting the sample rehydration solution into the tray; (b) placing the IPG strip with dried gel side down onto the fluid; (c) pipetting a few milliliters of paraffin oil on the IPG strip support film; and (d) closing the tray with the lid. These ceramic trays are accessories of the IPGphor.
Figure M11.8 Tray for cup loading and running IPG gel strips facing up. This tray is an accessory of the IPGphor.
Figure M11.9 Pipetting the agarose sealing solution and insertion of the equilibrated IPG strip into the SDS gel cassette. Marker proteins are pipetted on sample applicators and placed on the gel edge before applying the agarose.
Figure M11.10 Placing two equilibrated IPG strips on a standard-size SDS gel. Example of a horizontal chamber: Multiphor. The electrode positions are continuously variable. Dotted lines on the electrode wicks indicate the positions where the electrodes should be placed.
Figure M11.11 One equilibrated long IPG strip on a large SDS gel. The gel has the size of the cooling plate of the Multiphor chamber. To accommodate electrode wicks and electrodes, electrode wicks are cut into 2.5-cm-wide strips, and stacks of two strips are applied on each side. In the Multiphor, the electrode positions are continuously variable. Dotted lines on the electrode wicks indicate the positions where the electrodes should be placed.
Figure M11.12 Generating a uniform layer of cooling contact fluid by distributing the fluid evenly.
Figure M11.13 Soaking the electrode wicks with the respective buffer in the PaperPool. Air bubbles are rolled out.
Figure M11.14 Equipment for readymade HPE™ gels. (a) Single-chamber Blue Horizon; (b) multiple chamber Blue Tower for up to four gels. Electrodes can be adjusted to three different distances.
Figure M11.15 Device for simultaneous staining of up to five gels.
Figure M11.16 Two-dimensional electrophoresis of 150 µg E. coli extract. IEF in 24 cm IPG strip pH 4–7, cup loading at the anode end. SDS-PAGE in 0.65-mm-thick homogeneous polyacrylamide gel with 12.5%T on nonfluorescent film backing. Fluorescent staining with ServaPurple. Scanned with Typhoon multifluorescence imager with a green laser at 532 nm; emission filter 610 nm bandpass 30.

Method 12: PAGE of DNA Fragments

Figure M12.1 Preparation of the slot-former for short (a) and long (b) separation distances.
Figure M12.2 Applying the support film with a roller.
Figure M12.3 Assembling the gel cassette.
Figure M12.4 Pouring the polymerization solution.
Figure M12.6 Soaking the wicks with electrode buffer. Roll out the air bubbles.
Figure M12.7 Appliance for short- and long-distance runs of DNA fragments. Example for a horizontal chamber: Multiphor. Dotted lines on the electrode wicks indicate the positions where the electrodes should be placed.
Figure M12.8 Separation of DNA fragments in a rehydrated polyacrylamide gel with a discontinuous buffer system. Silver staining is used.

List of Tables

Chapter 1: Electrophoresis

Table 1.1 Comparison of flatbed and vertical gel systems

Method 3: Titration Curve Analysis

Table M3.1 Composition for monomer solution for gels with T = 4.2%

Method 4: Native PAGE in Amphoteric-Buffers

Table M4.1 Composition of the polymerization solutions
Table M4.2 Rehydration solution (0.6 mol l⁻¹ HEPES)
Table M4.3 Power supply program

Method 5: Agarose IEF

Table M5.1 Electrode solutions for IEF in agarose gels

Method 6: PAGIEF in Rehydrated Gels

Table M6.1 Power supply program for PAGIEF (polyacrylamide gel isoelectric focussing)
Table M6.2 Ammoniacal silver staining protocol (250 ml per step for manual, 150 ml per step for automated staining)
Table M6.3 Electrode solutions for IEF in polyacrylamide gels

Method 7: Horizontal SDS-PAGE

Table M7.1 Composition of the monomer solutions for two gradient gels 8–20%T and a sample application plateau with 5%T
Table M7.2 Catalyst volumes
Table M7.3 Programmable power supply
Table M7.4 Silver staining according to Heukeshoven and Dernick (1985)

Method 8: Vertical PAGE

Table M8.1 Composition of the monomer solutions for two discontinuous gels
Table M8.2 Composition of the gel solutions for two gradient gels
Table M8.3 Composition of the gel solutions for 12 discontinuous gels
Table M8.4 Composition of the gel solutions for 12 gradient gels
Table M8.5 Gel solutions for two gels for the analysis of small peptides
Table M8.6 Gel solutions for one blue native gel (example for gel size 18 cm × 24 cm, 1 mm)

Method 10: IEF in Immobilized pH Gradients

Table M10.1 Narrow pH gradients: Immobiline quantities for 15 ml of the starting solutions (2 gels); 0.2 mol l⁻¹ Immobiline in microliters
Table M10.2 Wide pH gradients: Immobiline quantities for 15 ml of the starting solutions (2 gels); 0.2 mol l⁻¹ Immobiline in microliters
Table M10.3 Recipes for starting solutions for two 0.5-mm-thick IPG gels pH 4–10, T = 4%, C = 3%
Table M10.4 Rehydration of Immobiline gels

Method 11: High-Resolution 2D Electrophoresis

Table M11.1 Examples of excitation light wavelengths and emission filter selection for imaging DIGE gels
Table M11.2 Composition of the polymerization solutions for IPG gels
Table M11.3 Pipetting volumes for the gradient mixer (Mix. ch. = Mixing chamber)
Table M11.4 Composition of three gel solutions for a gradient 12–15%T and a plateau with 6%T
Table M11.5 Pipetting volumes for the gradient mixer (Mix. ch. = Mixing chamber)
Table M11.6 Power supply settings for IPG-IEF on conventional equipment
Table M11.7 Running conditions for 11- and 24-cm IPG strips (at 20 °C)
Table M11.8 Silver staining according to Heukeshoven and Dernick (1985)

Method 12: PAGE of DNA Fragments

Table M12.1 Composition of the gel solutions for one gel
Table M12.2 Long-distance run in a 10%T gel, Tris–phosphate/Tris–borate buffer, 15 °C
Table M12.3 Ramping program. Volt level: gradient
Table M12.4 Sensitive silver staining (20–50 pg per band)

Appendix: Troubleshooting

Table A.1 Gel characteristics
Table A.2 Problems during IEF
Table A.3 Separations
Table A.8 Gel properties
Table A.9 Effects during washing
Table A.10 Effects during drying
Table A.11 Effects during rehydration
Table A.12 Effects during the IEF run
Table A.13 Separation results
Table A.14 Specific staining problems with IPG gels
Table A.15 Gel casting
Table A.16 Effects during electrophoresis
Table A.17 Separation results
Table A.18 SDS-PAGE specific staining problems
Table A.19 Drying
Table A.20 Gel preparation
Table A.21 Running the gels
Table A.22 Separation results
Table A.23 Preservation
Table A.24 First dimension: IEF in IPG strips
Table A.25 Second dimension: horizontal SDS-electrophoresis
Table A.26 DIGE fluorescent labeling
Table A.27 Assembling the blotting sandwich
Table A.28 Problems during blotting
Table A.29 After the transfer
Table A.30 Results
Table A.31 Detection
Table A.32 Preparing the samples
Table A.33 Running the gels
Table A.34 Silver staining

Author

Dr.-Ing. Reiner Westermeier
Auenstraße 4a
85354 Freising
Germany

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Foreword

The number of electrophoretic separation methods has increased dramatically since Tiselius' pioneering work, for which he received the Nobel Prize. Development of these methods has progressed from paper, cellulose acetate membranes and starch gel electrophoresis to molecular sieve, disc, SDS and immunoelectrophoresis and, finally, to isoelectric focussing but also to high-resolution two-dimensional electrophoresis. Together with silver and gold staining, autoradiography, fluorography, and blotting, these techniques afford better resolution, sensitivity and specificity for the analysis of proteins. In addition, gel electrophoresis has proved to be a unique method for DNA sequencing, while high-resolution two-dimensional electrophoresis has smoothed the fascinating path from isolation of the protein to the gene through amino acid sequencing and, after gene cloning, to protein synthesis.

The spectrum of analytical possibilities has become so varied that an overview of electrophoretic separation methods seems desirable not only for beginners but also for experienced users. This book has been written for this purpose.

The author belongs to the circle of the bluefingers, and experienced this in Milan in 1979 when he was accused of being a money forger when buying cigarettes in a kiosk after work because his hands were stained by Coomassie. Prof Righetti and I had to extricate him from this tricky situation. According to Maurer's definition (Proceedings of the first small conference of the bluefingers, Tübingen 1972), an expert was at work on this book and he can teach the whitefingers who only know of the methods by hearsay, for example, how not to get blue fingers.

As it is, I am sure that this complete survey of the methods will help not only the whitefingers but also the community of the bluefingers, silverfingers, goldfingers, and so on, and will teach them many technical details.

February 1990
Prof Dr Angelika Görg
Weihenstephan
Freising-Weihenstephan
FG Proteomik, Technische Universität München

Abbreviations, Symbols, Units

2D electrophoresis	two-dimensional electrophoresis
A	ampere
acc.	according
A,C,G,T	adenine, cytosine, guanine, thymine
ACES	N-2-acetamido-2-aminoethanesulfonic acid
AEBSF	aminoethyl benzylsulfonyl fluoride
AFLP	amplified restriction fragment length polymorphism
API	atmospheric pressure ionization
APS	ammonium persulfate
ARDRA	amplified ribosomal DNA restriction analysis
AU	absorbance units
16-BAC	benzyldimethyl-n-hexadecylammonium chloride
BAC	bisacryloylcystamine
Bis	N,N′-methylenebisacrylamide
BNE	blue native electrophoresis
bp	base pair
BSA	bovine serum albumin
C	crosslinking factor (%)
CA	carbonic anhydrase
CAF	chemically assisted fragmentation
CAM	coanalytical modification
CAPS	3-(cyclohexylamino)-propanesulfonic acid
CCD	charge-coupled device
CHAPS	3-(3-cholamidopropyl)dimethylammonio-1-propane sulfonate
CE	capillary electrophoresis
CID	collision induced dissociation
conc.	concentrated
CM	carboxylmethyl
CN-PAGE	clear native page
const.	constant
CTAB	cetyltrimethylammonium bromide
Da	dalton
DAF	DNA amplification fingerprinting
DBM	diazobenzyloxymethyl
DEA	diethanolamine
DEAE	diethylaminoethyl
DGGE	denaturing gradient gel electrophoresis
DHB	2,5-dihydroxybenzoic acid
DIGE	difference gel electrophoresis
Disc	discontinuous
DMSO	dimethylsulfoxide
DNA	desoxyribonucleic acid
DPT	diazophenylthioether
dsDNA	double stranded DNA
DSCP	double strand conformation polymorphism
DTE	dithioerythritol
DTT	dithiothreitol
E	field strength in volt per centimeter
EDTA	ethylenediaminetetraacetic acid
ESI	electro spray ionization
EST	expressed sequence tag
FT-ICR	Fourier transform – ion cyclotron resonance
GC	group specific component
GMP	good manufacturing practice
h	hour
HED	hydroxyethyldisulfide
HEPES	N-2-hydroxyethylpiperazine-N′-2-ethananesulfonic acid
HMW	high molecular weight
HPCE	high performance capillary electrophoresis
HPLC	high performance liquid chromatography
I	current in ampere, milliampere
ICPL	isotope-coded protein labeling
IEF	isoelectric focusing
IgG	immunoglobulin G
IPG	immobilized pH gradients
ITP	isotachophoresis
kB	kilobases
kDa	kilodaltons
K_R	retardation coefficient
LED	light emitting diode
LIF	laser induced fluorescence
LMW	low molecular weight
M	mass
mA	milliampere
MALDI	matrix assisted laser desorption ionization
MCE	microchip electrophoresis
MEKC	micellar electrokinetic chromatography
MES	2-(N-morpholino)ethanesulfonic acid
min	minute
mol/L	molecular mass
MOPS	3-(N-morpholino)propanesulfonic acid
m_r	relative electrophoretic mobility
mRNA	messenger RNA
MS	mass spectrometry
Msⁿ	mass spectrometry with n mass analysis experiments
MS/MS	tandem mass spectrometry
MW	molecular weight
NAP	nucleic acid purifier
Nonidet	nonionic detergent
NEPHGE	non equilibrium pH gradient electrophoresis
NHS	N-hydroxy-succinimide
O.D.	optical density
P	power in watt
p.a.	per analysis
PAG	polyacrylamide gel
PAGE	polyacrylamide gel electrophoresis
PAGIEF	polyacrylamide gel isoelectric focusing
PBS	phosphate buffered saline
PCR	polymerase chain reaction
PEG	polyethylene glycol
PFG	pulsed field gel (electrophoresis)
PGM	phosphoglucose mutase
pI	isoelectric point
PI	protease inhibitor
pK	dissociation constant
PMSF	phenylmethyl-sulfonyl fluoride
PPA	piperidino propionamide
PSD	postsource dissociation (decay)
PTM	posttranslational modification
PVC	polyvinylchloride
PVDF	polyvinylidene difluoride
r	molecular radius
RAPD	random amplified polymorphic DNA
REN	rapid efficient nonradioactive
Rf	value relative distance of migration
RFLP	restriction fragment length polymorphism
R_m	relative electrophoretic mobility
RNA	ribonucleic acid
RPA	ribonuclease protection assay
RuBP	ruthenium II tris-bathophenantroline disulfonate
s	second
SDS	sodium dodecyl sulfate
SNP	single nucleotide polymorphism
ssDNA	single stranded DNA
T	total acrylamide concentration [%]
TBE	tris borate EDTA
TBP	tributyl phosphine
TBS	tris buffered saline
TCA	trichloroacetic acid
TCEP	tris(2-carboxyethyl)phosphine
TEMED	N,N,N′,N′-tetramethylethylenediamine
TF	transferrin
TGGE	temperature gradient gel electrophoresis
ToF	time of flight
Tricine	N,tris(hydroxymethyl)-methyl glycine
Tris	tris(hydroxymethyl)-aminoethane
U	voltage in volt
V	volume in liter
v	speed of migation in meter per second
v/v	volume per volume
VLDL	very low density lipoproteins
W	watt
WiFi	wireless local area network (artificial abbreviation)
w/v	weight per volume (mass concentration)
ZE	zone electrophoresis

Preface

German Version

This book was written for the practitioner of electrophoresis in the laboratory. For this reason, we have avoided physico-chemical derivations and formulas concerning electrophoretic phenomena.

The type of explanation and presentation comes from several years of experience in giving user seminars and courses, writing handbooks, and solving user problems. They should be clear for technical assistants as well as for researchers in the laboratory. The commentary column offers room for personal notes.

In Part I, an introduction – as short as possible – to the actual state of the art is given. The references are not meant to be exhaustive.

Part II contains exact instructions for 11 chosen electrophoretic methods that can be carried out with one single piece of equipment. The sequence of the methods was planned so that an electrophoresis course for beginners and advanced users can be established afterwards. The major methods used in biology, biochemistry, medicine, and food science have been covered.

If – despite following the method precisely – unexplained effects should arise, their cause and remedies can be found in the troubleshooting guide in the Appendix.

The author would welcome any additional comments and solutions for the troubleshooting guide that the reader can supply.

Freiburg, March 1990
R. Westermeier

English Version, Fifth Edition

More than a decade has passed since the last update of this book. In the meantime, new methods have been developed in all areas of electrophoresis, workflows have been simplified, sensitivity of detection has been improved, and more experience has been added. Therefore it was high time to bring out a new, revised edition. Many lecture tours, congresses, and hands-on workshops on proteomics and electrophoresis techniques inspired me to change the order of the chapters and update information in all sections. Since the book Proteomics in Practice had been published in a new edition, and new mass spectrometry methodologies have been evolved, a special chapter on proteomics was no longer needed. Furthermore, as many DNA typing methods are now performed with alternative and more automated techniques, this part could be shortened.

Freising, August 2015
R. Westermeier

Electrophoresis in Practice

A Guide to Methods and Applications of DNA and Protein Separations

Foreword

Abbreviations, Symbols, Units

Preface

German Version

English Version, Fifth Edition