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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.

Detection of Blotted Proteins: Methods and Protocols

1. Western Blotting: Origin and Ascent of the Species

W. Neal Burnette

2. Methods to Concentrate Proteins for Protein Isolation, Proteomic and Peptidomic Evaluation

J.P. Dean Goldring

3. Measuring Protein Concentration on Nitrocellulose and After the Electrophoretic Transfer of Protein to Nitrocellulose

J.P. Dean Goldring

4. Detection of Blotted Proteins: Not all Blockers are Created Equal

Vishal Kothari and Suresh T. Mathews

5. Protein Stains to Detect Antigen on Membranes

Anil D'souza and Hal Scofield

6. Fluorescent Labeling of Proteins and Its Application to SDS-PAGE and Western Blotting

F. Javier Alba and Salvador Bartolomé

7. Rapid, Antibody-free Detection of Recombinant Proteins on Blots Using Enzyme Fragment Complementation

Neil W. Charter, Joe Horecka, Chin-Yee Loh, Albert Doan, Tom Wehrman, and Keith R. Olson

8. Use of Non-Radioactive Detection Method for North- and South-Western Blot

Claudia Franke, Daniel Gräfe, Holger Bartsch, Michael Bachmann

9. Immunoblotting using Radiolabeled Reagents for Detection

Holger Bartsch, Claudia Franke, Michael Bachmann

10. Immunoblotting of Antigens: Whole, Strip and New-Line Nitrocellulose Membrane Immunoblotting Using the Chemiluminescence Technique

Yaser Dorri

11. Detection of Protein Carbonyls by Means of Biotin Hydrazide -Streptavidin Affinity Methods

Kenneth Hensley

12. Direct Immunodetection of Antigens within the Precast Polyacrylamide Gel

Surbhi Desai, Boguslawa R. Dworecki, and Marie C. Nlend

13. Quantitative Analysis of Signal Transduction with In-Cell Western Immunofluorescence Assays

Vince Boveia and Amy Schutz-Geschwender

14. Ultrasensitive Protein Detection on Dot Blots and Western Blots with Semiconducting Polymer Dots

Fangmao Ye, Polina B Smith, and Daniel T. Chiu

15. Co-detection of Target and Total Protein by CyDye Labeling and Fluorescent ECL-plex Immunoblotting in a Standard Proteomics Workflow

Caitriona Scaife, Ciara A. McManus, Pamela M. Donoghue, and Michael J. Dunn

16. Using Biotinylated Proteins to Demonstrate Immunodetection of Antigens via Western Blotting, Dot blots and Immunohistochemistry

Thomas Millar, Ronald Knighton and Jo-Anne Chuck

17. Calcium Binding by Ro 60 Multiple Antigenic Peptides on PVDF Membrane

Biji T. Kurien and Michael Bachmann

18. Sequential use of Immunoblots for Characterization of Autoantibody Specificities

Holger Bartsch and Michael Bachmann

19. Nanogold Immunodetection Detection Systems for the Identification of Autoantigens by Western Blotting

Jacen S. Moore and R. Hal Scofield

20. Application of Intermittent Microwave Irradiation to Western Blot Analysis

Yu-Ting Liu and Shinya Toyokuni

21. Visualization of Unstained Protein Bands on PVDF

Jun Park, Masaharu Mabuchi, and Ajay Sharma

22. Multiplexed Fluorescent Immunodetection Using Low Auto-Fluorescence Immobilon®-FL Membrane

Jun Park, Masaharu Mabuchi, and Ajay Sharma

23. Cold Microwave-Enabled Protein Detection and Quantification

Niels Grützner, Romy M. Heilmann, Aaron G. Smith, Carol B. Johnson, Stanislav Vitha, Jörg M. Steiner, and Andreas K. Holzenburg

24. TLC-Blot (Far-Eastern Blot) and Its Application to Functional Lipidomics

Takao Taki

25. Analysis of Electroblotted Proteins by Mass Spectrometry

Jose L. Luque-Garcia and Thomas A. Neubert

26. On Membrane Renaturation of Recombinant Ro60 Autoantigen by Calcium Ions

Biji T. Kurien and Michael P. Bachmann

27. Phosphoprotein Detection on Protein Electroblot Using a Phosphate-Specific Fluorophore

Lee Broderick Bockus and R. Hal Scofield

28. Purification of Tryptic Digests on Polyvinylidene Difluoride Membrane

Biji T. Kurien and R. Hal Scofield

29. Detection of Blotted Proteins on Nitrocellulose/PVDF Membranes by Alta

Jayanta K. Pal, Shilpa J. Rao, and Dhanashri J. Godbole

30. Non-stripping "Rainbow" and Multiple Antigen Detection (MAD) Western Blotting

Stan (Stanislaw) Krajewski, Michelle M. Tsukamoto, Xianshu Huang, and Sebastian B. Krajewski

31. Supported Molecular Matrix Electrophoresis

Yu-ki Matsuno and Akihiko Kameyama

32. Parafilm-M^®, an Available Cost Effective Alternative for Immuno-blot Pouches

Syed MS Quadri

33. Succinylation-Alcian Blue Staining of Mucins on Polyvinylidene Difluoride Membranes

Akihiko Kameyama, Weijie Dong, and Yu-ki Matsuno

34. Comparison of Chemiluminescence vs. Infrared Techniques for Detection of Fetuin-A in Saliva

Suresh T. Mathews, Emily Graff, Robert L. Judd, and Vishal Kothari, M.Pharm

35. A Novel Methodology for Stripping and Reprobing of Western Blots Originally Developed with Colorimetric Substrate TMB

Parmita Kar, Saurabh Kumar Agnihotri, Archana Sharma, Rekha Sachan , and Madan Lal Bhatt, Monika Sachdev

36. Other Notable Methods of Membrane Protein Detection: A Brief Review

Biji T. Kurien and R. Hal Scofield

37. Nitrocellulose Membrane: The New Canvas

Jasmin R. Kurien and Bianca A Kurien

38. Invisible Ink Marking in ECL Membrane Assays

Biji T. Kurien