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AAT Bioquest

Buccutite™ Rapid APC-Cy5.5 Tandem Antibody Labeling Kit *Microscale Optimized for Labeling 25 ug Antibody Per Reaction*

APC-Cy5.5 is a popular color used in flow cytometry. Its primary absorption peak is at 651 nm with emission peak at~700 nm. AAT Bioquest offers this Buccutite™ rapid labeling kit to facilitate the APC-Cy5.5 tandem conjugations to antibodies and other proteins such as streptavidin and other secondary reagents. Buccutite™ APC-Cy5.5 Tandem Conjugation Kit provides a robust and convenient method to conjugate antibodies with APC-Cy5.5 tandem. The kit includes a preactivated APC-Cy5.5 tandem and reaction buffer. The entire process only requires two simple mixings without further purification required. The conjugated antibody can be used in flow cytometry, WB, ELISA and IHC applications. This kit is sufficient for 2 labeling reactions, each up to 25 ug of antibody. The best ratio for any new antibody reagent must be determined by experimentation. Our kit provides preactivated APC-Cy5.5 tandem to facilitate the APC-Cy5.5 tandem conjugations to antibodies and other proteins such as streptavidin and other secondary reagents. Our preactivated APC-Cy5.5 tandem is ready to conjugate, giving much higher yield than the conventionally tedious SMCC-based conjugation chemistry. In addition, our preactivated APC-Cy5.5 tandem is conjugated to a protein via its amino group that is abundant in proteins while SMCC chemistry targets the thiol group that has to be regenerated by the reduction of antibodies.

Example protocol

AT A GLANCE

Protocol Summary
  1. Add 1.25 µL Reaction Buffer (Component C) into antibody (25 µL)
  2. Add 2.5 µL Buccutite™ MTA working solution
  3. Incubate at room temperature for 30 - 60 minutes
  4. Mix with 50 µL Buccutite™ FOL-Activated APC-Cy5.5 working solution
  5. Incubate at room temperature for 60 minutes
Important Note

Upon receipt, store the kit at 4 °C. When stored properly, the kit should be stable for six months. Alternatively Components A and B can be stored at -20 °C. Do not freeze Reaction Buffer (Component C). Warm all the components and centrifuge the vials briefly before opening, and immediately prepare the required solutions before starting your conjugation. The following SOP is an example for labeling goat anti-mouse IgG antibody.

PREPARATION OF WORKING SOLUTION

Antibody working solution

For labeling 25 µg antibody (assuming the target antibody concentration is 1 mg/mL), mix 1.25 µL (5% of the total reaction volume) of Reaction Buffer (Component C) with 25 µL of the target antibody solution.

Note: If you have a different concentration, adjust the antibody volume accordingly to make ~25 µg antibody available for your labeling reaction.

Note: The antibody should be dissolved in 1X phosphate buffered saline (PBS), pH 7.2-7.4; If the antibody is dissolved in glycine buffer, it must be dialyzed against 1X PBS, pH 7.2-7.4, or use Amicon Ultra-0.5, Ultracel-10 Membrane, 10 kDa ( Cat. # UFC501008 from Millipore) to remove free amines or ammonium salts (such as ammonium sulfate and ammonium acetate) that are widely used for antibody precipitation.

Note: Impure antibodies or antibodies stabilized with bovine serum albumin (BSA) or gelatin will not be labeled well.

Note: The antibody –Buccutite™ MTA reaction efficiency is significantly reduced if the antibody concentration is less than 1 mg/mL. For optimal labeling efficiency the final antibody concentration range of 1-10 mg/mL is recommended.

Buccutite ™ MTA working solution

Add 10 µL DMSO (Not provided) into the vial of Buccutite ™ MTA (Component B).

Buccutite ™ FOL-Activated APC-Cy5.5 working solution

Add 50 µL ddH2O into the vial of Buccutite ™ FOL-Activated APC-Cy5.5 (Component A).

SAMPLE EXPERIMENTAL PROTOCOL

Run Antibody-Buccutite™ MTA reaction
  1. Add 2.5 µL of Buccutite ™ MTA working solution into antibody working solution, and mix them well by repeatedly pipetting for a few times or vortex the vial for a few seconds.
  2. Keep the antibody- Buccutite ™ MTA reaction mixture at room temperature for 30 - 60 minutes.

    Note: The antibody-Buccutite™ MTA reaction mixture can be rotated or shaken for longer time if desired.

Make Antibody-APC-Cy5.5 conjugation
  1. Add 50 µL of Buccutite™ FOL-Activated APC-Cy5.5 working solution with Antibody-Buccutite™ MTA solution, mix well by repeatedly pipetting for a few times or vortex the vial for a few seconds.
  2. Incubate for 1 -2 hours.
  3. The antibody-APC-Cy5.5 conjugate is now ready to use.

    Note: For immediate use, the antibody-APC-Cy5.5 conjugate need be diluted with the buffer of your choice.

    Note: For longer term storage, antibody-Cy5.5 conjugate solution need be concentrated or freeze dried.

Storage of Antibody-APC-Cy5.5 Conjugate

The antibody conjugate should be stored in the presence of a carrier protein (e.g., 0.1% bovine serum albumin) and 0.02-0.05% sodium azide. The Ab-APC-Cy5.5 conjugate solution could be stored at 4 °C for two months without significant change and kept from light.

Table 1. Available fluorophores at AAT Bioquest Buccutite™ Rapid Antibody Labelling Kits

Cat# Labels Ex (nm) Em (nm)
1312 PE 565 575
1340 PE-Cy5 565 674
1341 PE-Cy5.5 565 700
1342 PE-Cy7 565 780
1343 PE-Texas Red 565 600
1313 APC 651 662
1347

APC-iFluor® 700

651 713
1350 APC-Cy5.5 651 700
1351 APC-Cy7 651 780
1353 PerCP 482 677

1348

APC-iFluor® 750

651

791

Spectrum

References

View all 46 references: Citation Explorer
Chromophore attachment to phycobiliprotein beta-subunits: phycocyanobilin:cysteine-beta84 phycobiliprotein lyase activity of CpeS-like protein from Anabaena Sp. PCC7120
Authors: Zhao KH, Su P, Li J, Tu JM, Zhou M, Bubenzer C, Scheer H.
Journal: J Biol Chem (2006): 8573
Excitation energy transfer from phycobiliprotein to chlorophyll d in intact cells of Acaryochloris marina studied by time- and wavelength-resolved fluorescence spectroscopy
Authors: Petrasek Z, Schmitt FJ, Theiss C, Huyer J, Chen M, Larkum A, Eichler HJ, Kemnitz K, Eckert HJ.
Journal: Photochem Photobiol Sci (2005): 1016
Single-molecule spectroscopy selectively probes donor and acceptor chromophores in the phycobiliprotein allophycocyanin
Authors: Loos D, Cotlet M, De Schryver F, Habuchi S, Hofkens J.
Journal: Biophys J (2004): 2598
Isolation and characterisation of phycobiliprotein rich mutant of cyanobacterium Synechocystis sp
Authors: Prasanna R, Dhar DW, Dominic TK, Tiwari ON, Singh PK.
Journal: Acta Biol Hung (2003): 113
Evaluation of Tolypothrix germplasm for phycobiliprotein content
Authors: Prasanna R, Prasanna BM, Mohammadi SA, Singh PK.
Journal: Folia Microbiol (Praha) (2003): 59
Page updated on December 1, 2024

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Catalog Number1350
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Spectral properties

Extinction coefficient (cm -1 M -1)

700000

Excitation (nm)

651

Emission (nm)

700

Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12171501

Components

AAT Bioquest offers this Buccutite™ rapid labeling kit to facilitate the APC-Cy5.5 tandem conjugations to antibodies and other proteins such as streptavidin and other secondary reagents.  Our preactivated APC was premodified with our Buccutite™ FOL. Your antibody (or other proteins) is modified with our Buccutite™ MTA to give MTA-modified protein (such as antibody). The MTA-modified protein readily reacts with FOL-modified APC to give the desired APC-antibody conjugate in much higher yield than the SMCC chemistry. In addition, our preactivated APC reacts with MTA-modified biopolymers at much lower concentrations than the SMCC chemistry.
AAT Bioquest offers this Buccutite™ rapid labeling kit to facilitate the APC-Cy5.5 tandem conjugations to antibodies and other proteins such as streptavidin and other secondary reagents.  Our preactivated APC was premodified with our Buccutite™ FOL. Your antibody (or other proteins) is modified with our Buccutite™ MTA to give MTA-modified protein (such as antibody). The MTA-modified protein readily reacts with FOL-modified APC to give the desired APC-antibody conjugate in much higher yield than the SMCC chemistry. In addition, our preactivated APC reacts with MTA-modified biopolymers at much lower concentrations than the SMCC chemistry.
AAT Bioquest offers this Buccutite™ rapid labeling kit to facilitate the APC-Cy5.5 tandem conjugations to antibodies and other proteins such as streptavidin and other secondary reagents.  Our preactivated APC was premodified with our Buccutite™ FOL. Your antibody (or other proteins) is modified with our Buccutite™ MTA to give MTA-modified protein (such as antibody). The MTA-modified protein readily reacts with FOL-modified APC to give the desired APC-antibody conjugate in much higher yield than the SMCC chemistry. In addition, our preactivated APC reacts with MTA-modified biopolymers at much lower concentrations than the SMCC chemistry.