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Buccutite™ Rapid PerCP Antibody Labeling Kit *Microscale Optimized for Labeling 100 ug Antibody Per Reaction*

AAT Bioquest offers this Buccutite™ rapid labeling kit to facilitate the PerCP conjugations to antibodies and other proteins such as streptavidin and other secondary reagents.  Our preactivated PerCP 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 PerCP to give the desired PerCP -antibody conjugate in much higher yield than the SMCC chemistry. In addition, our preactivated PerCP reacts with MTA-modified biopolymers at much lower concentrations than the SMCC chemistry.
AAT Bioquest offers this Buccutite™ rapid labeling kit to facilitate the PerCP conjugations to antibodies and other proteins such as streptavidin and other secondary reagents.  Our preactivated PerCP 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 PerCP to give the desired PerCP -antibody conjugate in much higher yield than the SMCC chemistry. In addition, our preactivated PerCP reacts with MTA-modified biopolymers at much lower concentrations than the SMCC chemistry.
Ordering information
Price ()
Catalog Number1325
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)350000
Excitation (nm)477
Emission (nm)678
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)
350000
Excitation (nm)
477
Emission (nm)
678
PerCP (Peridinin-chlorophyll-protein complex) is isolated from Dinophyceae sp. It has an extremely high extinction coefficient, a high quantum efficiency and a large Stokes shift. It is well excited with the Argon laser at 488 nm with its maximum emission peak at 677 nm. AAT Bioquest offers this Buccutite™ rapid labeling kit to facilitate the PerCP conjugations to antibodies and other proteins such as streptavidin and other secondary reagents. Buccutite™ PerCP Conjugation Kit provides a robust and convenient method to conjugate antibodies with PerCP. The kit includes a preactivated PerCP and reaction buffer. 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 100 ug of antibody. The best ratio for any new antibody reagent must be determined by experimentation. Our kit provides preactivated PerCP to facilitate the PerCP conjugations to antibodies and other proteins such as streptavidin and other secondary reagents. Our preactivated PerCP is ready to conjugate, giving much higher yield than the conventionally tedious SMCC-based conjugation chemistry. In addition, our preactivated PerCP 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 PerCP2 vials (lyophilized)
Component B: Buccutite™ MTA2 vials (lyophilized)
Component C: Reaction Buffer1 Vial (20 µL)

Example protocol


AT A GLANCE

Protocol Summary
  1. Add 5 µL Reaction Buffer (Component C) into antibody (100 µL)
  2. Add the antibody solution into Buccutite™ MTA vial (Component B)
  3. Incubate at room temperature for 30 minutes
  4. Mix with 50 µL Buccutite™ FOL-Activated PerCP (Component A)
  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, Component B can be stored at -20 °C. Do not freeze Buccutite™ FOL-Activated PerCP (Component A), 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 100 µg antibody (assuming the target antibody concentration is 1 mg/mL), mix 5 µL (5% of the total reaction volume) of Reaction Buffer (Component C) with 100 µL of the target antibody solution.
Note     If you have a different concentration, adjust the antibody volume accordingly to make ~100 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 ReadiUse™ 10KD Spin Filter (Cat. # 60502 from AAT Bioquest) 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.

SAMPLE EXPERIMENTAL PROTOCOL

Run Antibody-Buccutite™ MTA reaction
  1. Add the antibody working solution directly into the vial of Buccutite ™ MTA (Component B), 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-PerCP conjugation
  1. Make Buccutite™ FOL-Activated PerCP solution by adding 25 µL ddH2O into the vial of Buccutite™ FOL-Activated PerCP (Component A), mix well by repeatedly pipetting for a few times or vortex the vial for a few seconds.
  2. Mix whole vial of Buccutite™ FOL-Activated PerCP solution into the antibody-Buccutite™ MTA solution, mix well and rotating the mixture for 1 hour at room temperature.
  3. The antibody-PerCP conjugate is now ready to use.
    Note     For immediate use, the antibody-PerCP conjugate need be diluted with the buffer of your choice. 

Storage of Antibody-PerCP Conjugate
The antibody conjugate should be stored at > 0.5 mg/mL in the presence of a carrier protein (e.g., 0.1% bovine serum albumin). The Antibody-PerCP conjugate solution could be stored at 4 °C for two months without significant change when stored in the presence of 2 mM sodium azide and kept from light. For longer storage, the antibody-PE conjugates could be lyophilized and stored at ≤ –20 °C.
Table 1.Available fluorophores at AAT Bioquest Buccutite™ Rapid Antibody Labelling Kits
Cat# Labels Ex (nm) Em (nm)
1310 PE 565 575
1322 PE-Cy5 565 674
1316 PE-Cy5.5 565 700
1317 PE-Cy7 565 780
1318 PE-Texas Red 565 600
1311 APC 651 662
1319 APC-iFluor™ 700 651 713
1320 APC-Cy5.5 651 700
1321 APC-Cy7 651 780
1325 PerCP 482 677

Spectrum


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spectrum

Spectral properties

Extinction coefficient (cm -1 M -1)350000
Excitation (nm)477
Emission (nm)678

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