Prelude: You are free to copy and distribute these documents at will--but please do so in their entirety, complete with figures. To reference this material, please include the WWW address , shown below, as well as the latest modification date: August, 2004, plus any references listed with the detailed conjugation protocol that you use.
Typical reference: M. Roederer, Conjugation of monoclonal antibodies (August,
Address any questions, comments, or suggestions to Mario Roederer.
In this series of
web pages, protocols, notes, and various illustrations are given to aid in the
conjugation of proteins--principally monoclonal antibodies--to fluorescent
dyes. These conjugation procedures are commonly performed in our laboratory--we
have conjugated several hundred different monoclonals using almost all of the
various dyes listed. The procedures are relatively straightforward and require
only minimal familiarity with standard laboratory techniques (gel filtration
and spectrophotometry are the most difficult!).
Virtually every protocol is designed for the conjugation of IgG (see below for conjugating other proteins). Most of these protocols will work just fine with any isotype--even IgM--or any Ig from any animal species. However, the reductive cross-linking procedures, used for conjugation of phycobiliproteins (PE and APC), their tandem derivatives, and Texas Red, may not work well for some IgM antibodies. Nonetheless, it is probably worth a try--we have had stunning success conjugating IgM antibodies with PE, APC, and Cy7APC.
The conjugation of immunoglobulins is very straight-forward--any laboratory should be able to make these procedures routine. For conjugation of PE and APC, however, the raw material can be expensive when purchased in the small quantities needed for conjugation. If you intend to conjugate many antibodies (e.g., 100 mg of various antibodies), consider buying the phycobiliproteins in bulk (1 gram lots) from a vendor. If you are only conjugating small amounts, it is worthwhile considering the relatively inexpensive conjugation kits available from ProZyme, Inc.--these kits come with all of the materials you need to prepare PE or APC conjugates, and let you perform the entire conjugation in 2 hours. These conjugation kits were developed based on the protocols described here. Other conjugations (like FITC, Cascade Blue, etc.) require relatively inexpensive raw materials--allowing you to optimize the conjugations in-house without great expense.
There are also protocols given for conjugation of these fluorescent
molecules to Annexin V. These protocols can serve as a models for the
fluorescent conjugation of nearly any protein.
No protocol is given for antibody purification after conjugation (e.g., Protein A or Protein G). In general, we do not purify our conjugates directed against cell-surface antigens, since unreacted dye is removed during the washing steps. Our conjugates generally have low background levels. However, you may find it useful to purify conjugates, especially if they are to be used for intracellular (cytoplasmic) staining.
The conjugations fall into four basic protocols: Type 1 (used for FITC, Cy5, and the initial preparation of the Cy5 and Cy7 tandem dyes); Type 2 (used for Biotin and Cascade Blue); Type 3 (used for conjugation of PE, APC, TR-BSA, and their derivatives); and Type 4 (used for the initial preparation of non-immunoglobulin proteins like Annexin V). Buffers for all Type 1 reactions are identical, as for Type 2, etc.; some buffers are identical across the different reaction protocols.
Procedures for the conjugation of immunoglobulins are provided for the following molecules:
In addition, protocols for conjugation of Annexin V to the following molecules are given as models for fluorescent conjugation of non-immunoglobulin proteins. Conjugation of other fluorescent molecules would proceed very similarly to the protocols listed below (use as a template the protocol Type that is appropriate for the molecule you are interested int).
You may view the fluorescence spectra of the fluorescent molecules listed above (all together), or view individual fluorescence spectra for:
You can also view absorbance spectra for:
Many people have contributed to the successful conjugations that we routinely perform in the laboratory. In particular, Aaron Kantor designed and optimized many of the basic protocols referenced in these pages, and made the chemical conjugations simple for all of the biologists here. Randy Hardy and Alan Stall devised the original reductive cross-linking protocols used for all the protein-protein conjugations. Other people who have been helpful in the development of the more recent protocols include Masahiko Amano, Michael Anderson, Stephen DeRosa, Rachel Gerstein, Peter Katsikis, Gina Jager, and Iwan Tjioe.
These pages were designed and written by Mario Roederer.