Invitrogen™ SiteClick™ R-PE DIBO

Why use R-PE DIBO reagents for antibody labeling?

R-Phycoerythrin combines high fluorescence intensity with broad visible-light excitation, making it a widely used label for sensitive cell analysis. The DIBO-modified R-PE reagent has a molar extinction coefficient of approximately 1,960,000 M-1 cm-1 at 565 nm, supporting strong signal generation from R-PE-labeled antibodies.

DIBO functionalization enables a copper-free click reaction with azide-modified antibodies under mild conditions. When the antibody is modified through SiteClick chemistry, the reactive azide groups are introduced on Fc-associated glycans, helping position the R-PE label away from the Fab antigen-binding domains.

Key benefits include:

• Site-selective, covalent attachment of R-PE to azide-modified IgG antibodies
• Helps preserve access to antigen-binding domains by directing labeling to Fc-associated glycans
• Bright red-orange fluorescence for sensitive flow cytometry and fluorescence-based detection
• Copper-free reaction conditions that avoid harsh antibody reduction steps
• More controlled and reproducible labeling than random amine- or thiol-reactive approaches
• Simple conjugation workflow with only a few minutes of hands-on setup followed by overnight incubation

Designed for SiteClick antibody-modification workflows

SiteClick technology takes advantage of conserved carbohydrate structures on the antibody heavy-chain Fc domain. The SiteClick™ Quartet and SiteClick™ Duet Antibody Modification Kits can be used to generate azide-modified antibodies that are ready to react with the DIBO-modified R-PE label. The shorter protocol for SiteClick Duet is often preferred. Because modification occurs away from the antigen-binding region, the workflow is designed to maintain antibody recognition while providing a defined site for label attachment.

How to conjugate R-PE DIBO to an antibody

Begin with approximately 100 micrograms of purified, azide-modified IgG. If the antibody concentration is below approximately 0.75 mg/mL or the sample is in a buffer containing azide, concentrate and buffer-exchange the antibody before starting the conjugation reaction. The user guide describes 1X Tris, pH 7.0, for this optional preparation step.

For the 100 microgram reaction, use 100-125 microliters of azide-modified antibody at approximately 0.75 mg/mL and add 75 microliters of R-PE DIBO in a 1.5 mL centrifuge tube. Vortex the reaction mixture, briefly centrifuge, and incubate in the dark for 20-24 hours at 20-25°C to allow the copper-free click reaction to proceed.

After conjugation, store the R-PE-labeled antibody at 2-8°C protected from light. Do not freeze. For long-term storage, sodium azide or thimerosal may be added, if appropriate for the downstream application.

Biological applications

SiteClick R-PE DIBO antibody conjugates are especially well suited for:

• Flow cytometry and multiparameter cell analysis
• Cell-surface antigen and biomarker detection
• Quantitative fluorescence measurements requiring a bright reporter
• Fluorescence microscopy and immunocytochemistry where R-PE detection is appropriate
• Multiplex immunoassays, microarrays, and other fluorescence-based binding assays

R-PE can be excited efficiently across several visible wavelengths and emits strongly near 578 nm, making it compatible with commonly used blue, green, and yellow flow-cytometer excitation lines. Its high brightness is particularly valuable when detecting low-abundance targets or when strong signal separation is needed in multicolor experiments.