Abstract
Immunolocalization of antigen via fluorescence requires that fluorochromes be linked either to the primary antibody (direct method) or to a second antibody (indirect method) to provide a fluorescent signal to mark the site of antibody-antigen binding. Of these two methods, the indirect technique is generally more useful and practical. Fluorochromes can be covalently conjugated to antibodies through reactions with thiol or amine groups. Typically, fluorochromes containing isothiocyanate, succinimidyl ester, or sulfonyl chloride reactive groups are conjugated to amines on the antibody molecules. Provided are step-by-step instructions for conjugating isothiocyanate derivates of fluorescein and sulfonyl chloride derivatives of rhodamine to the amine groups of antibodies.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Coons AH, Creech HJ, Jones RN (1941) Immunological properties of an antibody containing a fluorescent group. Proc Soc Exp Biol Med 47:200–202
Coons AH, Leduc EH, Connolly JM (1955) Studies on antibody production I. A method for the histochemical demonstration of specific antibody and its application to a study of the hyperimmune rabbit. J Exp Med 102:49–60
Mullins JM (1999) Fluorochromes: Prope-rties and characteristics. Methods Mol Biol
Titus JA, Haugland R, Sharrow SO, Segal DM (1982) Texas Red, a hydrophilic, red-emitting fluorophore for use with fluorescein in dual parameter flow microfluorometric and fluorescence microscopic studies. J Immunol Methods 50:193–204
Oi VT, Glazer AN, Stryer L (1982) Fluorescent phycobiliprotein conjugates for analyses of cells and molecules. J Cell Biol 93:981–986
Bochner BS, McKelvey AA, Schleimer RP, Hildreth JE, MacGlashan DW Jr (1989) Flow cytometric methods for the analysis of human basophil surface antigens and viability. J Immunol Methods 125:265–271
Panchuk-Voloshina N, Haugland RP, Bishop-Stewart J, Bhalgat MK, Millard PJ, Mao F, Leung WY, Haugland RP (1999) Alexa dyes, a series of new fluorescent dyes that yield exceptionally bright, photostable conjugates. J Histochem Cytochem 47:1179–1188
Schreiber AB, Haimovich J (1983) QuantiÂtative fluorometric assay for detection and characterization of Fc receptors. Methods Enzymol 93:147–155
Jaiswal JK, Simon SM (2004) Potentials and pitfalls of fluorescent quantum dots for biological imaging. Trends Cell Biol 14:497–504
Jovin TM (2003) Quantum dots finally come of age. Nat Biotechnol 21:32–33
Wu X, Liu H, Liu J, Haley KN, Treadway JA, Larson JP, Ge N, Peale F, Bruchez MP (2003) Immunofluorescent labeling of cancer marker Her2 and other cellular targets with semiconductor quantum dots. Nat Biotechnol 21:41–46
Jaiswal JK, Mattousi H, Mauro JM, Simon SM (2003) Long-term multiple color imaging of live cells using quantum dot bioconjugates. Nat Biotechnol 21:47–21
Hua X-F, Liu T-C, Cao Y-C, Liu B, Wang H-Q, Wang J-H, Huang Z-L, Zhao Y-D (2006) Characterization of the coupling of quantum dots and immunoglobulin antibodies. Anal Bioanal Chem 386:1665–1671
Susumu K, Uyeda HT, Medintz I, Pons T, Delehanty JB, Mattoussi H (2007) Enhancing the stability and biological functionalities of quantum dots via compact multifunctional ligands. J Am Chem Soc 129:13987–13996
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Mao, SY., Mullins, J.M. (2010). Conjugation of Fluorochromes to Antibodies. In: Oliver, C., Jamur, M. (eds) Immunocytochemical Methods and Protocols. Methods in Molecular Biology, vol 588. Humana Press. https://doi.org/10.1007/978-1-59745-324-0_6
Download citation
DOI: https://doi.org/10.1007/978-1-59745-324-0_6
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-58829-463-0
Online ISBN: 978-1-59745-324-0
eBook Packages: Springer Protocols