ReadiUse™ Preactivated APC-iFluor® 700 Tandem
| Overview |  SDSProtocol |
See also: PE and APC, Bioconjugation
Molecular weight 105000 | Extinction coefficient (cm -1 M -1) 700000 | Excitation (nm) 651 | Emission (nm) 710 |
Allophycocyanin (APC) is a phycobiliprotein isolated from Spirulina sp., a blue-green alga. Like other phycobiliproteins, APC is fluorescent, with an extremely high absorptivity and a high quantum efficiency. It is a protein which can be easily linked to antibodies and other proteins by conventional protein cross-linking techniques without altering its spectral characteristics. Our 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. ReadiUse™ Preactivated APC-iFluor® 700 Tandem is an activated APC protein, and can be easily conjugated to antibodies with much higher conjugation yield than the conventional APC.
Components
| A: ReadiUse™ Preactivated APC-iFluor™ 700 Tandem | 1 vial (1 mg) |
| B: Buccutite™ MTA | 1 vial (100 µg) |
| C: Spin Desalting Column | Not Included |
Example protocol
SAMPLE EXPERIMENTAL PROTOCOL
Preparation of pre-activated antibody with Buccutite™ MTA
- Reconstitute Buccutite™ MTA in DMSO at ~10 mg/mL.
Note Please store unused Buccutite™ MTA at -20 °C and could be used up to two freeze and thaw cycles. - Prepare target antibody (Ab) in pH = 8.5 - 9.0 buffer at concentration above 1 mg/mL.
- Add Buccutite™ MTA to Ab solution at the ratio of 8 - 10 µg Buccutite™ MTA/100 µg Ab.
- Mix well and react at room temperature for 60 minutes, rotating during the reaction.
- Purify the reaction mixture with desalting column to remove unreacted Buccutite™ MTA and exchange buffer to PBS or buffer of your choice.
- Collect the Buccutite™ MTA-activated Ab, and estimate the concentration by 70% yield of the original starting amount.
Conjugation with pre-activated APC-iFluor™ 700 Tandem
- Reconstitute pre-activated APC-iFluor™ 700 Tandem in 100 µL ddH2O to 10 mg/mL.
Note Reconstituted pre-activated APC-iFluor™ 700 Tandem could be stored at 4 °C for one month, kept from light. - Add APC-iFluor™ 700 Tandem directly to MTA-activated target Ab solution at the ratio of 130 µg APC-iFluor™ 700 Tandem/100 µg MTA-activated Ab.
- Rotate the mixture for 60 minutes at room temperature.
- The Ab/APC-iFluor™ 700 Tandem conjugates are now ready to use.
Note 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) and 0.02% - 0.05% sodium azide. The Ab/APC-iFluor™ 700 Tandem conjugates solution could be stored at 4 °C for up to two months, and kept from light. - (Optional) Ab/APC-iFluor™ 700 Tandem conjugates could be further purified through size exclusion chromatography to get best performance.
Calculators
Common stock solution preparation
Table 1. Volume of appropriate solvent needed to reconstitute specific mass of ReadiUse™ Preactivated APC-iFluor® 700 Tandem to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.
| 0.1 mg | 0.5 mg | 1 mg | 5 mg | 10 mg | |
| 1 mM | 952.381 nL | 4.762 µL | 9.524 µL | 47.619 µL | 95.238 µL |
| 5 mM | 190.476 nL | 952.381 nL | 1.905 µL | 9.524 µL | 19.048 µL |
| 10 mM | 95.238 nL | 476.19 nL | 952.381 nL | 4.762 µL | 9.524 µL |
Molarity calculator
Enter any two values (mass, volume, concentration) to calculate the third.
| Mass (Calculate) | Molecular weight | Volume (Calculate) | Concentration (Calculate) | Moles | ||||
| / | = | x | = |
Spectrum
Open in Advanced Spectrum Viewer
Spectral properties
| Extinction coefficient (cm -1 M -1) | 700000 |
| Excitation (nm) | 651 |
| Emission (nm) | 710 |
Product family
| Name | Excitation (nm) | Emission (nm) | Extinction coefficient (cm -1 M -1) |
| APC-iFluor® 700 Tandem | 685 | 710 | 700000 |
| PE-iFluor® 700 Tandem | 566 | 708 | 1960000 |
| APC-iFluor® 710 Tandem | - | - | - |
| APC-iFluor® 750 Tandem | 754 | 776 | 700000 |
| APC-iFluor® 800 Tandem | 804 | 660 | 700000 |
| APC-iFluor®A7 Tandem | - | - | - |
Images
Figure 1. Our preactivated APC-iFluor® 700 Tandem was premodified with our Buccutite™ FOL (provided). Your antibody (or other proteins) is modified with our Buccutite™ MTA (provided as free sample) to give MTA-modified protein (such as antibody). The MTA-modified protein readily reacts with FOL-modified APC-iFluor® 700 Tandem (provided) to give the desired APC-iFluor® 700 Tandem-antibody conjugate in much higher yield than the SMCC chemistry. In addition our preactivated APC-iFluor® 700 Tandem reacts with MTA-modified biopolymers at much lower concentrations than the SMCC chemistry.
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
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
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
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
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
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
Authors: Wu P., undefined
Journal: Sheng Li Ke Xue Jin Zhan (2000): 82