The Buccutite™ Antibody-Protein Conjugate Process.
Buccutite™ crosslinking technology provides an efficient method to conjugate proteins with another macromolecule such as an antibody
or an enzyme. More robust and with higher yield than the commonly-used SMCC, Buccutite™ crosslinking technology utilizes two exclusive linkers with unique properties. In general terms, these proprietary crosslinkers
work as two halves of a single homobifunctional crosslinker. Each has an amine-reactive group on one end which specifically targets primary amines on the desired protein. The other end contains our proprietary Buccutite™-reactive group, which has a high degree of affinity for only binding its respective Buccutite™ MTA/FOL crosslinker counterpart. When each is present, the two conjugates covalently link together at their Buccutite™-reactive site to form a protein-protein conjugate.
Standalone FOL and MTA Crosslinkers
Our Buccutite™ crosslinking technology provides the most convenient and effective crosslinking method to link two biomolecules with a high conjugation yield. Our method uses one pair of crosslinkers: Buccutite™ MTA and Buccutite™ FOL. MTA is added to one molecule, while FOL is added to another molecule. The cross-linking reaction is initiated by mixing Molecule-1-Buccutite™ MTA and Molecule-2-Buccutite™ FOL. This crosslinking reaction occurs under extremely mild and neutral conditions with no catalyst required. These standalone products allow for adaptation to the specific materials and requirements of the researcher.
We now offer the stand-alone Buccutite™ MTA and Buccutite™ FOL reagents to expand the application of Buccutite™ crosslinking technology. MTA is added to one molecule, while FOL is added to another molecule. The cross-linking reaction is initiated by mixing Molecule-1-Buccutite™ MTA and Molecule-2-Buccutite™ FOL. Conjugated reagent types include NHS esters, scavengers, and Dye 650.
Table 1. Buccutite™ FOL and MTA Products For Standalone FOL and MTA Crosslinkers
Peroxidase (HRP) Antibody Conjugation Kit
The Buccutite™ Peroxidase (HRP) Antibody Conjugation Kit is designed for preparing horseradish peroxidase (HRP)
conjugates directly from proteins, peptides, and other ligands that contain a free amino group. The HRP provided in our kit has been pre-activated with our proprietary linker Buccutite™ FOL, and can be directly used for conjugation. The entire process only requires two simple mixings without further purification required. The Buccutite™ FOL-activated HRP readily reacts with Buccutite™ MTA-containing molecules under extremely mild neutral conditions without a catalyst.
Table 2. Buccutite™ Peroxidase (HRP) Antibody Conjugation Kits For Peroxidase (HRP) Antibody Conjugation Kit
Rapid Labeling and Crosslinking Kits
Sample Antibody Rapid Labeling Protocol
Buccutite™ Rapid labeling and crosslinking kits provide a fast, simple and efficient technique for labeling PE, APC or tandem dyes to antibodies in microscale. It utilizes two separate linkers, Buccutite™ MTA and Buccutite™ FOL. These linkers are independently labeled to the phycobiliprotein and antibody of interest, and when mixed will bind strongly together resulting in an antibody-phycobiliprotein conjugate. Learn More
The simple process for Buccutite™ crosslinking technology for protein-protein conjugation.
Rapid Protein Crosslinking Kit
Using our proprietary pair of crosslinkers: Buccutite™ MTA and Buccutite™ FOL, the Buccutite™ Rapid Protein Crosslinking Kit
is designed to provide an easy method to produce high yields of conjugated antibodies. The efficient system can be simplified as follows: MTA is added to one protein, while FOL is added to another protein. Protein-protein cross-linking reaction is initiated by mixing Protein-1-Buccutite™ MTA and Protein-2-Buccutite™ FOL. If suitable non-serum-containing buffers
are used, purification requirements are minimal.
The mechanism of the Buccutite™ bioconjugation system used for the Rapid Protein Crosslinking Kit.
Rapid Antibody Labeling Kits
AAT Bioquest offers this Buccutite™ rapid labeling kit to facilitate the PE conjugations to antibodies and other proteins such as streptavidin and other secondary reagents. Buccutite™ Rapid Antibody Labeling Kits provide a robust and convenient method to conjugate your antibodies. The conjugated antibodies are suitable for use in WB, ELISA and IHC applications. Each kit provides preactivated APC or PE
to facilitate the conjugations to antibodies and other proteins such as streptavidin
and other secondary reagents
. Our preactivated material is ready to conjugate, giving much higher yield than the conventionally tedious SMCC-based conjugation chemistry. The kits include a reaction buffer and are sufficient for 2 labeling reactions each. The entire process only requires two simple mixings without further purification required. The best ratio for any new antibody reagent must be determined by experimentation, but each kit includes a protocol with recommended amounts for the user to adjust as necessary.
Table 3. Rapid Antibody Labeling Kits For Rapid Labeling and Crosslinking Kits
Rapid Tandem Antibody Labeling Kits
PE- and APC-Conjugated tandem dyes
are used frequently with flow cytometry
and other applications, as they are very useful for multiparameter visualization. Since tandem dyes can be excited by the same laser but give separated emission results, their use can improve experimental efficiency. All of the Buccutite™ Rapid Tandem Antibody Labeling Kits have identical excitation wavelengths of 575 nm for PE conjugates and 651 nm for APC conjugates, but a range of emission spectra, depending on each kits' secondary paired dye.
Table 4. PE-Conjugated Rapid Tandem Antibody Labeling Kits For Rapid Labeling and Crosslinking Kits
Table 5. APC-Conjugated Rapid Tandem Antibody Labeling Kits For Rapid Labeling and Crosslinking Kits
Available in 2 sizes, AAT Bioquest now offers our proprietary iFluor™ 700 dye
as an option for the APC-conjugated Buccutite™ Rapid Tandem Antibody Labeling Kit.
Table 6. APC-iFluor® 700 Rapid Tandem Antibody Labeling Kits For Rapid Labeling and Crosslinking Kits
AAT Bioquest's Custom Services
For very specific requirements, custom conjugations
are likely the most efficient solution.
- Guaranteed quality with 95% purity rating
- Affordable price tiers that work with your budget
- Scalable service with minimum order of 50 µg
- APC and PE custom conjugation in 3 working days. Other options available same-day.
- Supply your own protein or antibody or choose from 3000+ monoclonal and polyclonal antibodies in our catalog
- Antibody development also available
Other Series Using Buccutite™ Technology
The Buccutite™ conjugation technology's efficiency lends itself well to adaptation, and is represented in several other AAT Bioquest product series, either as part of the production process or part of the products' protocol.
Preactivated ReadiUse™ Single and Tandem Dyes
AAT Bioquest offers preactivated PE and APC
to facilitate conjugations to antibodies and other proteins such as streptavidin and other secondary reagents. Our preactivated ReadiUse™ dyes are ready to conjugate upon delivery and give much higher yield than traditional SMCC-based conjugation. In addition, our preactivated dyes bind to each target protein via its amino group that is abundant in proteins whereas SMCC chemistry targets the thiol group that has to be regenerated by the reduction of antibodies. Learn More
Table 7. ReadiUse™ Preactivated PE and APC Dyes For Other Series Using Buccutite™ Technology
Table 8. ReadiUse™ Preactivated PE and APC Tandem Dyes For Other Series Using Buccutite™ Technology
HRP-Labeled IgG Antibodies
Immunofluorescent image of paraffin-embedded human lung carcinoma labeled with Pan-Keratin Mouse mAb followed with HRP-labeled goat anti-mouse IgG (H+L) (Cat#16728
). The signal was developed with AAT's iFluor™ 488 tyramide
. Cells were also counterstained with DAPI
These highly purified and cross-absorbed purified IgGs are conjugated to HRP in high yield under a neutral condition to maximally retain the activities of both IgG and HRP using our proprietary Buccutite™ protein crosslinking technology.
Bucculite™ dU Incorporation Cell Proliferation Fluorescence Imaging Kit
Monitoring cell proliferation is one of the most reliable methods to assess cell viability, cell cycles and genotoxicity. A dependable way to detect cell proliferation is to measure DNA synthesis in the presence of thymidine during the S-phase of the cell cycle. Bucculite™ dU Incorporation Cell Proliferation Assay Kit
uses FOL-dU, an analog of thymidine. FOL-dU is incorporated into cellular DNA during DNA synthesis. After fixation, the incorporated FOL-dU is labelled with an MTA-iFluor™ conjugate through our Buccutite™ labeling technology. The resultant fluorophore-labeled DNA formed in cells is visualized in either the Cy5, TRITC, or FITC Channel, depending on which kit is used.
The Bucculite™ dU Incorporation Cell Proliferation Kit
provides an alternative to anti-BrdU antibody-based assay and EdU click chemistry assay. The assay is copper-free and environmentally-friendly, and extremely effective for measuring active DNA synthesis at single-cell level.
S-phase Hela cells were detected with Bucculite™ dU Incorporation Cell Proliferation Fluorescence Imaging Kit (Cat#22320
). HeLa cells at 50,000 cells/well/100 µL were seeded overnight in a 96-well black wall/clear bottom plate. Cells were treated with FOL-dU at 37ºC for 3 hours, and fixed with Methanol/PBS (90/10). After fixation, cells were stained with iFluor™ 647-MTA for 30min in staining buffer, and then washed three times with 1X washing Buffer. 100µL 5 µg/ml Hoechst 33342
solution in 1X Washing Buffer were added to each well and the fluorescence images were visualized with Cy5 filter for S phase cells (Red) and with DAPI filter nuclear for all cells (Blue).
Table 9. Bucculite™ dU Incorporation Cell Proliferation Fluorescence Imaging Kits For Other Series Using Buccutite™ Technology
Product Ordering Information
Table 10. Buccutite PE/APC Antibody Labeling Kits