Labeling Antibodies with IRDye800CW

IRDye800CW (IRDye800CW-N-hydroxysuccinimide ester, LI-COR Biosciences, Lincoln, NE) is a near infrared imaging probe with a broad absorption (778 nm) and emission (794 nm) peaks that is nontoxic to mice (18 (link)). Antibody conjugation results in an absorption of 774 nm and an emission of 789 nm (Biosciences L-C. IRDye(R) 800CW Protein Labeling Kit - High MW. Lincoln, NE: LI-COR Biosciences; 2007) (19 (link)). Control Immunoglobulin G (IgG), anti-rodent-VEGFR-2, and anti-rodent-Her2-Neu 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 hours at room temperature. Desalting spin columns (Pierce Biotechnology, Rockford, IL) removed the unconjugated dye. A final dye:protein ratio of 1.5 to 2.0 was determined using spectrophotometry.

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Morrison D.R., Sorace A.G., Hamilton E., Moore L.S., Houson H.A., Udayakumar N., Ovaitt A., Warram J.M, & Walsh E.M. (2021). Predicting Schwannoma Growth in a Tumor Model Using Targeted Imaging. Otology & neurotology : official publication of the American Otological Society, American Neurotology Society [and] European Academy of Otology and Neurotology, 42(5), e615-e623.

Publication 2021

Desalting spin columns

Antibodies Antibody Buffer Ester Her2 neu Immunoglobulin g Mice Potassium phosphate Protein R protein Rodent Spectrophotometry Vegfr 2

Corresponding Organization : University of Alabama

Other organizations : University of Alabama at Birmingham

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Variable analysis

independent variables

Antibody conjugation

dependent variables

Absorption wavelength of antibody-dye conjugate
Emission wavelength of antibody-dye conjugate

control variables

Incubation time (2 hours)
Incubation temperature (room temperature)
Potassium phosphate buffer (1.00 M, pH 9.0)
Dye:protein ratio (1.5 to 2.0)

controls

Positive control: Control Immunoglobulin G (IgG)
Negative controls: anti-rodent-VEGFR-2, anti-rodent-Her2-Neu

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