IRDye800CW (IRDye800CW-N-hydroxysuccinimide ester, LI-COR Biosciences, Lincoln, Nebraska) is nontoxic to mice.41 (link) IRDye800CW is a near infrared imaging probe with a broad absorption (778 nm) and emission (794 nm) peaks. Antibody conjugation results in an absorption of 774 nm and an emission of 789 nm.42 ,43 (link) Control IgG (Innovative Research, Peary Court Novi, MI) and panitumumab (Vectibix; Amgen, Thousands Oaks, California) were labeled according to the manufacturer’s instructions. Briefly, antibodies were incubated with IRDye800CW in 1.00 M potassium phosphate buffer (pH 9.0) for 2 hrs at room temperature. Desalting spin columns (Pierce Biotechnology, Rockford, IL) removed the unconjugated dye. A final dye:protein ratio of 1.5–2.0 was determined using spectrophotometry.
Protocol detail
Labeling Antibodies with IRDye800CW
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Antibodies
Antibody
Buffer
Ester
Mice
Oaks
Panitumumab
Potassium phosphate
Protein
Spectrophotometry
Vectibix
Corresponding Organization : University of Alabama at Birmingham
Other organizations : Stanford University
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- Conjugation of antibodies (control IgG, panitumumab) with IRDye800CW
- Absorption and emission peaks of the antibody-IRDye800CW conjugates
- Incubation of antibodies with IRDye800CW in 1.00 M potassium phosphate buffer (pH 9.0) for 2 hours at room temperature
- Removal of unconjugated dye using desalting spin columns
- Determination of final dye:protein ratio using spectrophotometry
- Positive control: Control IgG labeled with IRDye800CW
- Experimental group: Panitumumab labeled with IRDye800CW
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