Introduction to Antibodies

Chemicon International Inc. is pleased to offer the second edition of Introduction to Antibodies, a basic guide to immunological assays and general technical information. This is a handy reference to supplement those techniques described in the literature, recorded in general laboratory procedures, and described on individual product data sheets. As every antibody and experimental design is unique, these general assay suggestions should not be interpreted as applicable to all situations, but rather as an additional source of reference information describing techniques that have been successfully used for a variety of the antibodies available today. As always, individual assays must be optimized empirically and antibody titers must be established for every unique batch of antibody.

For more information about applications using any of Chemicon’s more than 7,000 antibodies and kits, Chemicon offers scientific advice for a variety of immunological applications via phone, fax or e-mail. In addition, the Chemicon website has extensive general and product-specific technical information available online and downloadable. We hope that you will contact us with any comments, questions, or suggestions that you may have.

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Table of Contents 

I. Introduction
	A. ANTIGENS B. ANTIBODIES C. ANTIBODY-ANTIGEN INTERACTION D. MONOCLONAL AND POLYCLONAL ANTIBODIES E. ANTIBODY FORMATS F. GENERAL TECHNICAL GUIDELINES
		1. Proper Controls 2. Handling of Reagents 3. Antibody and Titer
II. Antibody Applications
	A. IMMUNOPRECIPITATION
		1. Antigen Labeling 2. Sample Preparation 3. Formation of the Antibody-Antigen Complexes 4. Precipitation of Immune Complexes 5. Troubleshooting
	B. PROTEIN (WESTERN) BLOTTING
		1. Sample Preparation 2. Gel Electrophoresis 3. Membrane Transfer 4. Blocking Non-specific Binding 5. Addition of the Antibody 6. Detection 7. Troubleshooting
	C. ELISA
		1. Sandwich ELISA Protocol 2. Competitive ELISA Protocol 3. Troubleshooting
	D. IMMUNOHISTOCHEMISTRY / IMMUNOCYTOCHEMISTRY
		1. Specimen Preparation
			a. Fixatives b. Section Type
				i. Cryostat ii. Paraffin
		2. Antigen Retrieval
			a. Enzymatic Digestion b. Microwave c. Autoclaving or Pressure Cooking
		3. Antibody Staining 4. General Protocols for Immunohistochemical Staining 5. Antibody Detection
			a. Enzyme Mediated b. Fluorescence c. Signal Amplification
		6. Detection Substrates and Fluorescent Tables 7. Troubleshooting
			a. No Staining b. Weak Staining c. Background Staining
	E. FLOW CYTOMETRY
		1. Sample Protocol for Flow Cytometry
III. Appendix
	A. Fixative Recipes B. Serial Dilutions C. Protein A/G Binding Affinities D. ELISA Substrates E. Useful formulations for Western blotting and Immunoprecipitation F. Further Reading G. Glossary