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Western Blotting Principles and Methods

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Western Blotting Principles and Methods GE Healthcare Life Sciences Western Blotting Principles and Methods imagination at work Handbooks from GE Healthcare Life Sciences For more information refer to www.gelifesciences.com/handbooks GE Healthcare GE Healthcare GE Healthcare Life Sciences 2-D Electrophoresis using Life Sciences GST Gene Fusion System Life Sciences Multimodal immobilized pH gradients Handbook Chromatography Principles and Methods 18-1157-58 Handbook 80-6429-60 29-0548-08 2-D Electrophoresis Principles and Methods GST Gene Multimodal Fusion System Chromatography Handbook Handbook imagination at work GE Healthcare GE Healthcare GE Healthcare Life Sciences Affinity Chromatography Life Sciences High-throughput Process Life Sciences Nucleic Acid Sample Principles and Methods High-throughput Development with Preparation for Process Development with PreDictor™ Plates 18-1022-29 Principles and Methods PreDictor Plates Downstream Analyses Principles and Methods Principles and Methods Affinity Chromatography Nucleic Acid Sample Principles and Methods 28-9403-58 Preparation for 28-9624-00 Downstream Analyses Principles and Methods GE Healthcare GE Healthcare GE Healthcare Life Sciences ÄKTA Laboratory-scale Life Sciences Hydrophobic Interaction Life Sciences Protein Sample Chromatography Systems and Reversed Phase Preparation Instrument Management Chromatography Handbook Handbook Principles and Methods 28-9887-41 Hydrophobic Interaction and Reversed Phase 29-0108-31 11-0012-69 Protein Sample ÄKTA™ Laboratory-scale Chromatography Preparation Chromatography Systems Principles and Methods Handbook Instrument Management Handbook GE Healthcare GE Healthcare GE Healthcare Life Sciences Antibody Purification Life Sciences Imaging Life Sciences Purifying Challenging Handbook 473 532 635 650 685 785 Principles and Methods Proteins Laser UV IR epi 18-1037-46 trans 29-0203-01 Principles and Methods 312 CCD UV IR W 365 460 520 630 710 epi 28-9095-31 Purifying Imaging Challenging Proteins Principles and Methods Antibody Purification Principles and Methods Handbook GE Healthcare GE Healthcare GE Healthcare Life Sciences Biacore Assay Life Sciences Ion Exchange Life Sciences Recombinant Protein Biacore™ Assay Handbook Handbook Chromatography and Purification Handbook 29-0194-00 Chromatofocusing Principles and Methods Principles and Methods 18-1142-75 Ion Exchange 11-0004-21 Recombinant Chromatography & Chromatofocusing Protein Purification Principles and Methods Principles and Methods GE Healthcare GE Healthcare GE Healthcare Life Sciences Biacore Sensor Surface Life Sciences Isolation of Life Sciences Spectrophotometry Biacore BR-1005-71 mononuclear cells Handbook Sensor Surface Handbook Methodology and 29-0331-82 Applications Isolation of 18-1152-69 mononuclear cells Methodology and applications Spectrophotometry Handbook GE Healthcare Life Sciences MicroCal Calorimetry— GE Healthcare GE Healthcare Life Sciences Cell Separation Media Life Sciences Strategies for Protein MicroCal™ Calorimeters Achieving High Quality Methodology and Achieving high quality data Purification Data Applications Handbook Handbook 18-1115-69 28-9833-31 Cell Separation Media 29-0033-51 Methodology and applications Strategies for Protein Purif ication Handbook GE Healthcare Life Sciences Microcarrier Cell Culture GE Healthcare GE Healthcare Life Sciences Gel Filtration Life Sciences Western Blotting Principles and Methods Principles and Methods Principles and Methods 18-1140-62 18-1022-18 28-9998-97 Microcarrier Cell Culture Gel filtration Principles and Methods Principles and Methods Western Blotting Principles and Methods Western Blotting Principles and Methods Contents Chapter 1 - Introduction .....................................................................................................................9 Chapter 2 - Sample preparation .................................................................................................... 15 2.1 Introduction ...................................................................................................................................................15 2.2 Protein extraction options ......................................................................................................................16 2.2.1 Detergent-based lysis ...........................................................................................................16 2.2.2 Freeze/thaw lysis ....................................................................................................................16 2.2.3 Osmotic shock ..........................................................................................................................17 2.2.4 Ultrasonication ........................................................................................................................17 2.2.5 Mechanical methods ............................................................................................................17 2.2.6 Enzymatic digestion ..............................................................................................................17 2.2.7 Explosive decompression ..................................................................................................18 2.2.8 Premade lysis buffers and sample preparation kits...............................................18 2.2.9 Protecting your samples .....................................................................................................19 2.3 Sample cleanup ...........................................................................................................................................20 2.3.1 Sample cleanup products ...................................................................................................22 2.3.2 Depletion of high-abundance protein from serum or plasma samples .......23 2.3.3 Desalting and concentrating samples .........................................................................23 2.4 Determination of total protein concentration ...............................................................................23 2.4.1 Products for determination of total protein concentration ...............................25 2.5 References .....................................................................................................................................................25 Chapter 3 - Gel electrophoresis ..................................................................................................... 27 3.1 Electrophoresis ............................................................................................................................................27 3.1.1 Polyacrylamide gels...............................................................................................................28 3.1.2 Buffer systems and pH .........................................................................................................31 3.1.3 Denaturing gels: SDS-PAGE ................................................................................................32 3.1.4 Native gels: PAGE ....................................................................................................................33 3.1.5 Two-dimensional (2-D) gel electrophoresis ................................................................33 3.1.6 Electrophoresis equipment from GE Healthcare .....................................................34 3.2 Molecular weight markers .....................................................................................36 3.2.1 Rainbow Molecular Weight Markers .............................................................................37 3.2.2 Amersham ECL DualVue Western Blotting Markers ..............................................37 3.2.3 Amersham ECL Plex Fluorescent Rainbow Markers...............................................38 3.2.4 Unlabeled Protein Molecular Weight Markers ..........................................................39 3.3 Determining the molecular weight of unknown proteins from molecular weight markers .....................................................................................................................39 3.4 Total protein stains ....................................................................................................................................39 3.5 General polyacrylamide gel electrophoresis (PAGE) protocol ................................................41 3.6 References .....................................................................................................................................................42 Chapter 4 - Transfer ........................................................................................................................ 43 4.1 Protein transfer ............................................................................................................................................43 4.1.1 Electrotransfer .........................................................................................................................43 4.1.2 Diffusion transfer ....................................................................................................................50 4.2 Transfer buffers and running conditions .........................................................................................51 4.2.1 Notes on transfer of large and small proteins ..........................................................52 4.2.2 Current and transfer time ..................................................................................................53
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