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Buccutite™ Rapid APC-iFluor® 700 Tandem Antibody Labeling Kit *Microscale Optimized for Labeling 100 ug Antibody Per Reaction*
APC-iFluor® 700 Tandem is an excellent replacement for APC-Alexa Fluor® 700 Tandem since they have almost identical spectra. On some antibodies, our APC-iFluor® 700 Tandem is much brighter than APC-Alexa Fluor® 700 Tandem with a higher stain index. AAT Bioquest offers this Buccutite™ rapid labeling kit to facilitate the APC-iFluor® 700 tandem conjugations to antibodies and other proteins such as streptavidin and other secondary reagents. Buccutite™ APC-iFluor® 700 Tandem Conjugation Kit provides a robust and convenient method to conjugate antibodies with APC. The kit includes a preactivated APC-iFluor® 700 tandem 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 APC-iFluor® 700 tandem to facilitate the APC-iFluor® 700 tandem conjugations to antibodies and other proteins such as streptavidin and other secondary reagents. Our preactivated APC-iFluor® 700 tandem is ready to conjugate, giving much higher yield than the conventionally tedious SMCC-based conjugation chemistry. In addition, our preactivated APC-iFluor® 700 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
- Add 5 µl Reaction Buffer (Component C) into antibody (100 µl)
- Add the antibody solution into Buccutite™ MTA vial (Component B)
- Incubate at room temperature for 30 minutes
- Mix with 50 µL Buccutite™ FOL-Activated APC-iFluor™ 700 (Component A)
- Incubate at room temperature for 60 minutes
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
- 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.
- 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-iFluor™ 700 conjugation
- Make Buccutite™ FOL-Activated APC-iFluor™ 700 solution by adding 50 µL ddH2O into the vial of Buccutite™ FOL-Activated APC-iFluor™ 700 (Component A), mix well by repeatedly pipetting for a few times or vortex the vial for a few seconds.
- Mix whole vial of Buccutite™ FOL-Activated APC-iFluor™ 700 solution into the antibody-Buccutite™ MTA solution, mix well and rotating the mixture for 1 hour at room temperature.
- The antibody-APC-iFluor™ 700 conjugate is now ready to use.
Note For immediate use, the antibody-APC-iFluor™ 700 conjugate need be diluted with the buffer of your choice.
Storage of Antibody-APC-iFluor™ 700 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-APC-iFluor™ 700 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-APC-iFluor™ 700 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
Product family
| Name | Excitation (nm) | Emission (nm) | Extinction coefficient (cm -1 M -1) |
| Buccutite™ Rapid APC-iFluor® 750 Tandem Antibody Labeling Kit *Microscale Optimized for Labeling 25 ug Antibody Per Reaction* | 651 | 793 | 700000 |
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
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
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
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
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
Authors: Prasanna R, Prasanna BM, Mohammadi SA, Singh PK.
Journal: Folia Microbiol (Praha) (2003): 59
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