Allophycocyanin (APC) tandem conjugates combine the brightness and red laser compatibility of APC with far-red and near-infrared acceptor fluorophores, enabling expanded multicolor flow cytometry panels without requiring additional laser lines. Through fluorescence resonance energy transfer (FRET), these conjugates enable emission from acceptor fluorophores following APC excitation at its native 660 nm absorption maximum into the 700–820 nm spectral range, providing researchers with powerful tools for designing complex immunophenotyping experiments that expand available detection channels in multicolor panels.

AAT Bioquest offers APC tandems for a variety of applications, including conventional and spectral flow cytometry, with acceptors including classic and proprietary fluorescent dyes.

Allophycocyanin is a 105 kDa phycobiliprotein isolated from cyanobacteria (Spirulina sp.) and red algae, with excitation at 651 nm and emission at 660 nm. APC exhibits strong absorption at 633 nm and 647 nm red laser lines with a molar extinction coefficient of 700,000 cm⁻¹M⁻¹ and quantum yield of 0.68. While less bright than PE, APC's red laser excitation makes it an essential complement for multicolor panels, enabling additional detection channels without spectral crowding in the 488 nm laser range.

APC is the least stable of the major phycobiliproteins, susceptible to dissociation at low concentrations. Cross-linked APC (CL-APC), which is chemically stabilized between α and β subunits, maintains integrity during storage and dilute assay conditions. APC tandem conjugates leverage FRET to shift emission into NIR regions through coupling with cyanine dyes (Cy5.5, Cy7) or iFluor® acceptors, enabling emission wavelengths spanning 660–820 nm while maintaining red laser excitation.

The spectral properties table below provides excitation and emission maxima and filter configurations for each APC tandem conjugate. These specifications enable optimal instrument setup and facilitate panel design for multicolor flow cytometry experiments.

ReadiUse™ preactivated APC tandems are supplied in reactive formats ready for direct conjugation to antibodies and other proteins. These preactivated conjugates eliminate the need for separate activation chemistry, streamlining the labeling workflow while delivering higher conjugation yields than traditional approaches.

Amine-reactive APC tandems target primary amines (lysine residues and N-termini) present on antibodies and proteins. This chemistry provides straightforward conjugation without requiring antibody reduction, preserving disulfide bonds critical for maintaining antibody structure and binding activity. The amine-reactive format consistently achieves higher conjugation yields compared to thiol-reactive SMCC chemistry.

SMCC-activated APC tandems react specifically with free thiol groups generated by mild reduction of antibody disulfide bonds. This site-directed conjugation approach targets the hinge region of IgG antibodies, positioning the fluorophore away from antigen-binding sites. While requiring an additional reduction step, thiol-reactive chemistry offers controlled stoichiometry for applications demanding consistent labeling ratios.

Buccutite™ technology represents a significant advancement over conventional antibody labeling methods, achieving near 100% conjugation yield without purification through a proprietary click chemistry approach. The system employs two specialized crosslinkers—MTA and FOL—that react rapidly and specifically upon mixing, forming stable conjugates in under two hours.

Traditional SMCC-based APC conjugation requires reduction of antibody disulfide bonds to generate reactive thiols, a process that can increase the risk of antibody aggregation, loss of binding activity, and variable labeling results under suboptimal conditions. Buccutite™ chemistry eliminates these limitations by targeting native functional groups without disrupting antibody structure, preserving antigen recognition while ensuring complete conjugation efficiency.

The streamlined workflow requires only two incubation steps: addition of the MTA linker to the antibody (30 minutes) followed by addition of the FOL-activated APC tandem (60 minutes). The resulting conjugate is immediately ready for use in flow cytometry, immunofluorescence, or immunoassay applications without additional purification. Kits containing 1 mg of APC tandem include a spin column for optional buffer exchange and concentration.

For standard IgG antibodies (~150 kDa), typical reactions label one APC per antibody. While higher labeling ratios are achievable, excessive loading may reduce antigen binding due to steric hindrance at the antibody's variable regions.