-D Sample Prep + 2-D Electrophoresis + Imaging 2 Fourth Edition How-To Guide Positioning Guide Tray Positioning Stop Positioning the tray 18002 70994625354 1 2 3 4 5 6 1 7 2 8 3 9 10 4 11 5 12 6 7 8 9 10 11 12 3-10NL Metal Grounding Strip Positioning Guide 1 2 3 4 5 6 7 Location of Tray 8 9 10 11 2-D Electrophoresis Workflow Positioning Stop 12 Common Negative Pin Positioning Guide Instrument Platform 2-D Electrophoresis Guide Table of Contents Part I: Theory and Product Selection 5 Chapter 3 The First Dimension: Isoelectric Focusing (IEF) 33 Chapter 1 Overview of Protein Separation by Isoelectric Point (pI) 34 Two-Dimensional Electrophoresis 5 IEF Media: IPG Strips vs. Carrier Ampholytes 35 The Context of Proteomics 6 Selection of IPG Strips 36 Overview of Experimental Design 6 Choice of pH Gradient 36 Sample Preparation 8 Choice of IPG Strip Length 36 First-Dimension Separation: IEF 8 Second-Dimension Separation: SDS-PAGE 8 Estimation of pl 37 Detection 9 Image Acquisition, Analysis, and Spot Cutting 9 Sample Application 38 Protein Digestion and Identification by Mass Spectrometry 9 Sample Application during Rehydration 38 Sample Application by Cup Loading 39 Chapter 2 Sample Preparation 11 Setup for IEF 39 The Importance of Sample Preparation 12 Power Conditions for IEF 40 General Considerations 12 Chapter 4 The Second Dimension: Cell Lysis 12 SDS-PAGE 43 Protein Solubilization 15 Chaotropic Agents 15 Protein Separation by Size 44 Detergents 15 Reducing Agents 16 Selection of Polyacrylamide Gels 44 Ampholytes, Buffers, and Other Additives 17 Choice of Gel Percentage (Composition) 45 Choice of Gel Size 47 Removal of Interfering Substances 19 Choice of Buffer System 47 General Considerations 19 Nucleic acids (DNA and RNA) 19 Transition from First to Second Dimension 52 Polysaccharides 20 Power Conditions and Reagents for SDS-PAGE 52 Phenolic Compounds 20 Lipids 20 Molecular Weight Estimation 53 Salts and Other Small Ionic Compounds 20 Prevention of Keratin Contamination 21 Chapter 5 Detection 55 Prefractionation 22 Detection of Proteins in Gels 56 Fractionation by Subcellular Location 22 Coomassie Stains 56 Fractionation by Solubility/Hydrophobicity 24 Silver Stains 57 Fractionation by Protein Charge 24 Fluorescent Stains 57 Fractionation by pI 25 Negative Stains 57 Fractionation by Size (MW) 26 Stain-Free Technology 57 Depletion and Dynamic Range Reduction 27 About This Guide Detection of Proteins on Western Blots 61 This guide describes the experimental methods and tools used in 2-D Depletion 27 Dynamic Range Reduction 28 electrophoresis and proteomics research. It provides background information about technologies common to all proteomics studies as well as protocols and ProteoMiner Technology 29 advice you can use as a starting point for your studies. This guide also explains Sample Quantitation (Protein Assays) 30 how experimental conditions can be varied and interpreted to optimize your results and provides an extensive set of references that you can consult for more information. Since each sample, experimental approach, and objective is different, this guide offers ideas for developing customized protocols suitable for the analysis of your samples. Bio-Rad’s Proteomics Program From sample preparation to protein analysis, Bio-Rad’s tools provide you with choices in methodology, protocols, and products. Our informative 2-D Electrophoresis and Analysis Applications and Technologies web pages are a valuable resource with video tutorials, protocols, troubleshooting tips, and much more. To learn more, visit www.bio-rad.com/2DElectroAnalysis. 1 2-D Electrophoresis Guide Table of Contents Chapter 6 Image Acquisition, Analysis, Chapter 9 First-Dimension IEF Part III: Troubleshooting 111 and Spot Cutting 63 with IPG Strips 85 Isoelectric Focusing 112 Finding Protein Spots of Interest 64 Tips for IEF 86 SDS-PAGE 113 Image Acquisition 64 IPG Strip Rehydration and Sample Loading 86 Total Protein Staining 114 Image Analysis 65 Performing IEF 87 2-D Gel Evaluation 115 Image Optimization, Spot Detection, and Quantitation 66 IPG Strip Rehydration in Rehydration/Equilibration Trays Gel Comparison 66 Followed by IEF 87 Data Normalization 66 IEF with Gel-Side Up 87 Part IV: Appendices 125 Data Analysis and Reporting 67 IEF with Gel-Side Down 88 Cup Loading (IEF with Gel-Side Up) 88 Appendix A 126 Spot Cutting from 2-D Gels 67 IPG Strip Rehydration in the Focusing Tray Followed by IEF 89 Glossary 126 Chapter 7 Identification and IEF Programming Recommendations 90 Appendix B 130 References 130 Characterization of 2-D Protein Spots 69 Chapter 10 Second-Dimension SDS-PAGE 95 Related Bio-Rad Literature 132 Beyond Excision 70 Tips for SDS-PAGE 96 Appendix C 133 Ordering Information 133 Proteolytic Digestion 70 IPG Strip Equilibration 97 Washing 70 Reduction and Alkylation 70 Sealing IPG Strips onto SDS-PAGE Gels 98 In-Gel Proteolytic Digestion 70 SDS-PAGE 99 Identification by Mass Spectrometry 71 Peptide Mass Fingerprinting 72 101 Tandem Mass Spectrometry (MS/MS) 73 Chapter 11 Protein Detection Tips for Total Protein Staining 102 Establishment of 2-D Databases 73 Long-Term Storage of Stained Gels 102 Part II: Methods 75 Total Protein Staining 103 Bio-Safe Coomassie Stain 103 Chapter 8 Sample Preparation 75 Flamingo Fluorescent Gel Stain 103 Oriole Fluorescent Gel Stain 104 Tips for Sample Preparation 76 SYPRO Ruby Protein Gel Stain 104 Lysis (Cell Disruption) 76 Silver Stain Plus Kit 105 Protein Solubilization 76 107 Buffers and Solutions 77 Chapter 12 In-Gel Trypsin Digestion Tryptic Digestion Protocol 108 Cell Lysis and Protein Extraction Procedures 78 Reagents and Solutions 108 Suspension Cultured Human Cells 78 Destaining Gel Plugs from Silver-Stained Gels 108 Monolayer Cultured Human Cells 78 (Pre-Treatment) Mammalian Tissue 79 General Destaining Protocol 109 Microbial Cultures 79 Reduction and Alkylation Protocol 109 Plant Leaves 80 Digestion Protocol 109 Sample Cleanup 81 Extraction Protocol 109 Buffer Exchange (Desalting) 81 Sample Quantitation (RC DC Protein Assay) 82 Microfuge Tube Assay Protocol (1.5 ml) 82 2 3 2-D Electrophoresis Guide Theory and Product Selection PART I Theory and Product Selection CHAPTER 1 Overview of Two-Dimensional Electrophoresis 4 5 2-D Electrophoresis Guide Chapter 1: Overview of Two-Dimensional Electrophoresis Theory and Product Selection The Context of Proteomics Other common methods of proteome analysis involve Proteome analysis (proteomics) is the comprehensive the proteolytic digestion of sample proteins and the 2-D Electrophoresis Workflow analysis of proteins present in a sample and chromatographic separation of the resulting peptides representing a particular physiological state at a coupled directly to mass spectrometric analysis. Peptides are identified by referencing a database, particular point in time. The aim of proteomics is to Sample Preparation determine the presence, relative abundance, and and their proteins of origin are inferred. While these posttranslational modification state of a large fraction methods are largely automatable and provide an of the proteins in a sample (Wilkins et al. 1996). impressive depth of proteome coverage, some Prepare the protein at a concentration and in a solution suitable for IEF. Choose a method that maintains the native charge, Since proteins are directly involved in cellular structure, information is lost when analyzing protein fragments solubility, and relative abundance of proteins of interest. regulation, and metabolism, proteomics can often yield instead of intact proteins. The 2-D electrophoresis a more informative and accurate picture of the state of approach maintains proteins in their intact states and a living cell than can analysis of the genome or mRNA. enables the study of isoform distribution, which is not possible if the sample is proteolytically digested prior One of the greatest challenges of proteome analysis to separation. Since proteins can be selected through is the reproducible separation of complex protein First-Dimension Separation: IEF image analysis, mass spectrometry need be applied mixtures while retaining both qualitative and only to the proteins of interest. This is an important quantitative relationships. Many combinations of Separate proteins according to pI by IEF. Select the appropriate IPG consideration when access to instrumentation or the techniques can be used to separate and analyze strip length and pH gradient for the desired resolution and sample expense of mass spectrometric analysis is a limitation. proteins, but two-dimensional (2-D) electrophoresis load. Select appropriate sample loading and separation conditions. is uniquely powerful in its ability to separate hundreds The suitability of 2-D electrophoresis to proteome to thousands of products simultaneously (Choe and analysis is clear, but its applications also extend to Lee 2000). This technique uses two different biomarker detection, development of drug and other electrophoretic separations, isoelectric focusing (IEF) therapies, and optimization and development of and SDS-PAGE, to separate proteins according to protein purification strategies. Second-Dimension Separation: SDS-PAGE their isoelectric point (pI) and molecular weight. The identities of individual protein spots from the gel Overview of Experimental Design Separate proteins according to size by SDS-PAGE. can then be identified by mass spectrometry (MS) The general workflow in a 2-D electrophoresis Select the appropriate gel size and composition of their tryptic peptides. Together with computer-