Protein to Protein Conjugation

Buccutite™ crosslinking technology offers a truly unique and trouble-free method of labeling antibodies with another macromolecule, such as an enzyme or phycobiliprotein. Compared to conventional techniques for protein-protein conjugation, such as the labor intensive SMCC chemical labeling method, Buccutite™ conjugation is significantly more robust and less demanding. These kits allow optimal labeling of microgram or milligram amounts of antibodies in less than two hours, with no purification necessary and 100% recovery of antibody. With Buccutite™ labeling kits, routinely prepare covalently-labeled conjugates that can tolerate the rigorous handling and washing that is required in a typical immunoassay (e.g. ELISA, IHC and Western blot) or flow cytometry application. AAT Bioquest offers Buccutite™ antibody labeling kits for preparing horseradish peroxidase (HRP), alkaline phosphatase (AP), PE, APC, PerCP and tandem dye antibody conjugates.

Too busy to perform your own conjugation or having difficulty finding the right labeling kit? Not to worry AAT Bioquest's bioconjugation service can prepare the custom antibody conjugate you need.

Buccutite™ vs SMCC Conjugation At-A-Glance

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1. FC = flow cytometry; IF = immunofluorescence; IHC = immunohistochemistry; WB = Western blot; ELISA = enzyme-linked immunosorbent assay.

Buccutite™ HRP and Poly-HRP Antibody Labeling Kits

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Buccutite™ Peroxidase (HRP) Antibody Labeling Kits enable researchers to efficiently label primary antibodies with the detection enzyme horseradish peroxidase (HRP) in less than 2 hours. To streamline the process, the HRP provided in these kits are preactivated with our proprietary linker Buccutite™ FOL, and can be directly used for antibody conjugation. Simply activate the antibody or protein you wish to label with the Buccutite™ MTA linker provided in each kit, and mix together with the Buccutite™ FOL-activated HRP. The resulting enzyme labeled antibody conjugates are ideal for various immunoassay applications including ELISA, IHC and western blotting, and are highly stable for long-term storage. Buccutite™ Peroxidase Antibody Labeling Kits are available in three sizes optimized for labeling 25 µg, 100 µg and 1 mg of antibody per labeling reaction.



For difficult applications that require an additional level of sensitivity, we offer Buccutite™ Poly-HRP Antibody Labeling kits. These kits enable the direct labeling of antibodies to polymers of HRP, resulting in conjugates that deliver the highest sensitivity and signal-to-noise ratios. Poly-HRP conjugates are ideal for applications where the target of interest is in low-abundance or when sample volume is limited.

Key features of the Buccutite™ HRP and Poly-HRP Antibody Labeling Kits

• Can effectively and efficiently label antibody concentrations as low as 100 µg/mL (SMCC requires at least 1 mg/mL)
• Labeled antibodies are ready in under 2 hours, with as little as ~15 minutes hands on-time
• Simple two step mixing protocol reduces batch-to-batch variation
• No column purification step required
• 100% conjugation yield
• Conjugates stable for long-term storage (4 °C, 12 months)
• Conjugates can be used for ELISA, IHC, blotting, Power Styramide Signal Amplification and TSA Imaging
• Offers flexibility in experimental design, HRP conjugates are compatible with colorimetric, fluorimetric and chemiluminescent substrates

High-performance alternative to Lightning-Link™ HRP Labeling Kit

Buccutite™ Rapid PE, APC, PerCP and Tandem Dye Antibody Labeling Kits

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Phycobiliproteins, such as phycoerythrin (PE), allophycocyanin (APC) and peridinin-chlorophyll-protein (PerCP), have the capacity to generate fluorescence signals significantly brighter than any commercially available synthetic fluorophore. When conjugated to molecules having biological specificity, such as antibodies, protein A or streptavidin, phycobiliproteins yield extraordinarily luminescent probes, that have found considerable success in fluorescence imgaging applications that require high sensitivity but not photostability, primarily flow cytometry, FACS and immunophenotyping. Moreover, phycobiliproteins can be labeled with small organic fluorophores to generate tandem dyes with exceptionally long Stoke's Shifts for multiparametric analysis using a single excitation source. For example, FITC, PE, PE-Cy5 and PE-Cy7 are ideal for multiparametric analysis since all four fluorophores can simultaneously be excited using the 488 nm laser line and they all demonstrate excellent spectral separation.



Buccutite™ Rapid Antibody Labeling Kits makes preparing antibodies for flow cytometry quick and easy. Using a simple two step mixing protocol, researchers can directly conjugate PE, APC, PerCP or a tandem dye to any antibody or protein in less than 2 hours. No column purification is necessary, so you recovery is always 100%. Buccutite™ Rapid Antibody Labeling Kits are available in three sizes optimized for labeling 25 µg, 100 µg or 1 mg of antibody per labeling reaction.

Key features of the Buccutite™ Rapid Antibody Labeling Kits

• Can effectively and efficiently label antibody concentrations as low as 100 µg/mL (SMCC requires at least 1 mg/mL)
• Labeled antibodies are ready in under 2 hours, with as little as ~15 minutes hands on-time
• Simple two step mixing protocol reduces batch-to-batch variation
• No column purification step required
• 100% conjugation yield
• Conjugates stable for long-term storage (4 °C, 12 months)
• Conjugtes ideal for multicolor flow cytometry applications

High-performance alternative to Lightning-Link™ Labeling Kits

Labeling Kit	CD4(SK3)- Conjugate	Stain Index
Buccutite™ Kit	CD4-PE Conjugate	55
Lightning-Link® Kit	CD4-PE Conjugate	28

Additional Resources

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Colorimetric, Fluorimetric and Chemiluminescent HRP Substrates

On it's own HRP conjugates have little value, as they lack the capacity to produce a measurable signal. To facilitate target visualization HRP conjugates require compounds known as substrates. In the presence of hydrogen peroxide, HRP catalyzes the oxidation of these substrates to produce a quantifiable signal. HRP substrates are commonly divided into three categories according to they type of singal it produces, these include, colorimetric, fluorimetric or chemiluminescent substrates.

• Colorimetric substrates produce a visible color-change that can be measured using an absorbance microplate reader.
• Fluorimetric substrates produce a highly fluorescent byproducts that can be measured using any fluorescence instrument. Offers greater sensitivity then colorimetric substrates.
• Chemiluminescent substrates generate luminescent byproducts that can be measured using a luminometer. Since the emission of a photon is a result of a chemical reaction and not light excitation, there is minimal background interference.