Detection of Blotted Proteins

This detailed volume provides methods and techniques for detection after blotting. Chapters guide readers through a number of variations on the theme of protein transfer to solid support followed by detection, presenting adaptations of traditional techniques, and original methods of protein blotting. Written for the Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Practical and authoritative, Detection of Blotted Proteins: Methods and Protocols presents numerous techniques based on the Western blot, providing detailed, readily reproducible methods, tips, and alternatives directly and easily transferable to the laboratory setting.

Includes cutting-edge methods and protocols Provides step-by-step detail essential for reproducible results Contains key notes and implementation advice from the experts

Inhalt

Western Blotting: Origin and Ascent of the Species.- Methods to Concentrate Proteins for Protein Isolation, Proteomic and Peptidomic Evaluation.- Measuring Protein Concentration on Nitrocellulose and After the Electrophoretic Transfer of Protein to Nitrocellulose.- Detection of Blotted Proteins: Not all Blockers are Created Equal.- Protein Stains to Detect Antigen on Membranes.- Fluorescent Labeling of Proteins and Its Application to SDS-PAGE and Western Blotting.- Rapid, Antibody-free Detection of Recombinant Proteins on Blots Using Enzyme Fragment Complementation.- Use of Non-Radioactive Detection Method for North- and South-Western Blot.- Immunoblotting using Radiolabeled Reagents for Detection.- Immunoblotting of Antigens: Whole, Strip and New-Line Nitrocellulose Membrane Immunoblotting Using the Chemiluminescence Technique.- Detection of Protein Carbonyls by Means of Biotin Hydrazide Streptavidin Affinity Methods.- Direct Immunodetection of Antigens within the Precast Polyacrylamide Gel.- Quantitative Analysis of Signal Transduction with In-Cell Western Immunofluorescence Assays.- Ultrasensitive Protein Detection on Dot Blots and Western Blots with Semiconducting Polymer Dots.- Co-detection of Target and Total Protein by CyDye Labeling and Fluorescent ECL-plex Immunoblotting in a Standard Proteomics Workflow.- Using Biotinylated Proteins to Demonstrate Immunodetection of Antigens via Western Blotting, Dot blots and Immunohistochemistry.- Calcium Binding by Ro 60 Multiple Antigenic Peptides on PVDF Membrane.- Sequential use of Immunoblots for Characterization of Autoantibody Specificities.- Nanogold Immunodetection DetectionSystems for the Identification of Autoantigens by Western Blotting.- Application of Intermittent Microwave Irradiation to Western Blot Analysis.- Visualization of Unstained Protein Bands on PVDF.- Multiplexed Fluorescent Immunodetection Using Low Auto-Fluorescence Immobilon®-FL Membrane.- Cold Microwave-Enabled Protein Detection and Quantification.- TLC-Blot (Far-Eastern Blot) and Its Application to Functional Lipidomics.- Analysis of Electroblotted Proteins by Mass Spectrometry.- On Membrane Renaturation of Recombinant Ro60 Autoantigen by Calcium Ions.- Phosphoprotein Detection on Protein Electroblot Using a Phosphate-Specific Fluorophore.- Purification of Tryptic Digests on Polyvinylidene Difluoride Membrane.- Detection of Blotted Proteins on Nitrocellulose/PVDF Membranes by Alta.- Non-stripping Rainbow and Multiple Antigen Detection (MAD) Western Blotting. - Supported Molecular Matrix Electrophoresis.- Parafilm-M ® , an Available Cost Effective Alternative for Immuno-blot Pouches.- Succinylation-Alcian Blue Staining of Mucins on Polyvinylidene Difluoride Membranes.- Comparison of Chemiluminescence vs. Infrared Techniques for Detection of Fetuin-A in Saliva.- A Novel Methodology for Stripping and Reprobing of Western Blots Originally Developed with Colorimetric Substrate TMB.- Other Notable Methods of Membrane Protein Detection: A Brief Review.- Nitrocellulose Membrane: The New Canvas.- Invisible Ink Marking in ECL Membrane Assays.