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

AAT Bioquest offers this Buccutite™ rapid labeling kit to facilitate the PE-Cy5.5 tandem conjugations to antibodies and other proteins such as streptavidin and other secondary reagents.  Our preactivated PE 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 PE to give the desired PE-antibody conjugate in much higher yield than the SMCC chemistry. In addition, our preactivated PE reacts with MTA-modified biopolymers at much lower concentrations than the SMCC chemistry.
AAT Bioquest offers this Buccutite™ rapid labeling kit to facilitate the PE-Cy5.5 tandem conjugations to antibodies and other proteins such as streptavidin and other secondary reagents.  Our preactivated PE 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 PE to give the desired PE-antibody conjugate in much higher yield than the SMCC chemistry. In addition, our preactivated PE reacts with MTA-modified biopolymers at much lower concentrations than the SMCC chemistry.
Ordering information
Price ()
Catalog Number1341
Unit Size
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Additional ordering information
Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Spectral properties
Extinction coefficient (cm -1 M -1)1960000
Excitation (nm)566
Emission (nm)671
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12171501

OverviewpdfSDSpdfProtocol


Extinction coefficient (cm -1 M -1)
1960000
Excitation (nm)
566
Emission (nm)
671
PE-Cy5.5 is a popular color used in flow cytometry. Its primary absorption peak is at 565 nm with emission peak at~700 nm. The filter sets of 682/33 nm and 695/40 nm are recommended for this tandem color. AAT Bioquest offers this Buccutite™ rapid labeling kit to facilitate the PE-Cy5.5 tandem conjugations to antibodies and other proteins such as streptavidin and other secondary reagents. Buccutite™ PE-Cy5.5 Conjugation Kit provides a robust and convenient method to conjugate your antibodies with PE. The kit includes a preactivated PE 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. Considering the large size of PE (240 kDa), the amount of antibody used in a labeling reaction must always be less than the amount of PE. The best ratio for any new antibody reagent must be determined by experimentation but 25 ug of IgG antibody for every 50 ug of PE usually gives optimal results. Our kit provides preactivated PE-Cy5.5 to facilitate the PE-Cy5.5 tandem conjugations to antibodies and other proteins such as streptavidin and other secondary reagents. Our preactivated PE-Cy5.5 tandem is ready to conjugate, giving much higher yield than the conventionally tedious SMCC-based conjugation chemistry. In addition, our preactivated PE-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.

Components


Component A: Buccutite™ FOL-Activated PE-Cy5.52 vials
Component B: Buccutite™ MTA1 vial
Component C: Reaction Buffer 1 vial (20 µL)

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 PE-Cy5.5 working solution
  5. Incubate at room temperature for 60 minutes 
Important      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

1. 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.


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

3. Buccutite ™ FOL-Activated PE-Cy5.5 working solution
Add 50 µL ddH2O into the vial of Buccutite ™ FOL-Activated PE-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-PE-Cy5.5 conjugation
  1. Add 50 µL of Buccutite™ FOL-Activated PE-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 to 2 hours.
  3. The antibody-PE-Cy5.5 conjugate is now ready to use.
    Note     For immediate use, the antibody-PE-Cy5.5 conjugate need be diluted with the buffer of your choice.
    Note     For longer term storage, antibody-PE-Cy5.5 conjugate solution need be concentrated or freeze dried. 

Storage of Antibody-PE-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-PE-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

Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Extinction coefficient (cm -1 M -1)1960000
Excitation (nm)566
Emission (nm)671

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
Co-ordinated expression of phycobiliprotein operons in the chromatically adapting cyanobacterium Calothrix PCC 7601: a role for RcaD and RcaG
Authors: Noubir S, Luque I, Ochoa de Alda JA, Perewoska I, T and eau de Marsac N, Cobley JG, Houmard J.
Journal: Mol Microbiol (2002): 749
Phycobiliprotein genes of the marine photosynthetic prokaryote Prochlorococcus: evidence for rapid evolution of genetic heterogeneity
Authors: Ting CS, Rocap G, King J, Chisholm SW.
Journal: Microbiology (2001): 3171
Phycobiliprotein-Fab conjugates as probes for single particle fluorescence imaging
Authors: Triantafilou K, Triantafilou M, Wilson KM.
Journal: Cytometry (2000): 226
Novel activity of a phycobiliprotein lyase: both the attachment of phycocyanobilin and the isomerization to phycoviolobilin are catalyzed by the proteins PecE and PecF encoded by the phycoerythrocyanin operon
Authors: Zhao KH, Deng MG, Zheng M, Zhou M, Parbel A, Storf M, Meyer M, Strohmann B, Scheer H.
Journal: FEBS Lett (2000): 9
Phycobiliprotein and fluorescence immunological assay
Authors: Wu P., undefined
Journal: Sheng Li Ke Xue Jin Zhan (2000): 82