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AAT Bioquest

Buccutite™ Rapid PE-Texas Red Tandem Antibody Labeling Kit *Microscale Optimized for Labeling 25 ug Antibody Per Reaction*

PE-Texas Red is a popular color used in flow cytometry. Its primary absorption peak is at 565 nm with emission peak at 600 nm. AAT Bioquest offers this Buccutite™ rapid labeling kit to facilitate the PE-Texas Red tandem conjugations to antibodies and other proteins such as streptavidin and other secondary reagents. Buccutite™ PE-Texas Red Conjugation Kit provides a robust and convenient method to conjugate your antibodies with PE-Texas Red tandem. The kit includes a preactivated PE-Texas Red 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. 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 RPE. The best ratio for any new antibody reagent must be determined by experimentation but 25 ug of IgG antibody for every 50 ug of RPE usually gives optimal results. Our kit provides preactivated PE-Texas Red to facilitate the PE-Texas Red tandem conjugations to antibodies and other proteins such as streptavidin and other secondary reagents. Our preactivated PE-Texas Red tandem is ready to conjugate, giving much higher yield than the conventionally tedious SMCC-based conjugation chemistry. In addition, our preactivated PE-Texas Red 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 PE-Texas Red 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

Antibody Working Solution
  1. To label 25 µg of 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 your antibody has a different concentration, adjust the volume to ensure approximately 100 µg of antibody is available for the 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: Ensure the antibody is pure and not stabilized with bovine serum albumin (BSA) or gelatin, as these stabilizers significantly impair labeling efficiency.

    Note: For optimal labeling efficiency, the antibody concentration should be in the range of 1–10 mg/mL. If the concentration is below 1 mg/mL, the Buccutite™ MTA reaction efficiency is significantly reduced.

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

Buccutite ™ FOL-Activated PE-Texas Red working solution
  1. Add 50 µL of ddH2O to the vial of Buccutite™ FOL-Activated PE-Texas Red (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-Texas Red conjugation
  1. Add 50 µL of Buccutite™ FOL-Activated PE-Texas Red 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-Texas Red conjugate is now ready to use.

    Note: For immediate use, the antibody-PE-Texas Red conjugate must be diluted with the buffer of your choice.

    Note: For longer term storage, antibody-PE-Texas Red conjugate solution must be concentrated or freeze dried.

Storage of Antibody-PE-Texas Red 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-Texas Red 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 November 27, 2024

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Catalog Number1343
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Spectral properties

Extinction coefficient (cm -1 M -1)

1960000

Excitation (nm)

565

Emission (nm)

613

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 PE-Texas Red 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-Texas Red 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-Texas Red 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.