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iFluor™ 647-streptavidin conjugate

HeLa cells were incubated with mouse anti-tubulin and biotin goat anti-mouse IgG followed by AAT’s iFluor™ 647-streptavidin conjugate (Red, Left) or streptavidin conjugated with Alexa Fluor<sup>®</sup> 647 (Red, Right), respectively.
HeLa cells were incubated with mouse anti-tubulin and biotin goat anti-mouse IgG followed by AAT’s iFluor™ 647-streptavidin conjugate (Red, Left) or streptavidin conjugated with Alexa Fluor<sup>®</sup> 647 (Red, Right), respectively.
Ordering information
Price ()
Catalog Number16966
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
Physical properties
Molecular weight52000
SolventWater
Spectral properties
Correction Factor (260 nm)0.03
Correction Factor (280 nm)0.03
Correction Factor (656 nm)0.0793
Extinction coefficient (cm -1 M -1)2500001
Excitation (nm)656
Emission (nm)670
Quantum yield0.251
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure
UNSPSC12352200
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Show More (43)

OverviewpdfSDSpdfProtocol


Molecular weight
52000
Correction Factor (260 nm)
0.03
Correction Factor (280 nm)
0.03
Correction Factor (656 nm)
0.0793
Extinction coefficient (cm -1 M -1)
2500001
Excitation (nm)
656
Emission (nm)
670
Quantum yield
0.251
AAT Bioquest's iFluor™ dyes are optimized for labeling proteins, in particular, antibodies. These dyes are bright, photostable and have minimal quenching on proteins. They can be well excited by the major laser lines of fluorescence instruments (e.g., 350, 405, 488, 555 and 633 nm). Streptavidin conjugates are widely used together with a conjugate of biotin for specific detection of a variety of proteins, protein motifs, nucleic acids and other molecules since streptavidin has a very high binding affinity for biotin. A variety of the complementary biotinylated reagents are available from numerous commercial vendors.This iFluor™ 647-streptavidin conjugate comprises streptavidin (as the biotin-binding protein) with iFluor™ 647 covalently attached (as the fluorescent label). It is commonly used as a second step reagent for indirect immunofluorescent staining, when used in conjunction with biotinylated primary antibodies. iFluor™ 647-streptavidin conjugates has fluorescence excitation and emission maxima of ~654 nm and ~674 nm respectively. These spectral characteristics make it an excellent alternative to Alexa Fluor® 647-streptavidin conjugate (Alexa Fluor® is the trademark of Invitrogen). It is a very valuable tool for biotin-streptavidin-based biological assays and tests.

Platform


Flow cytometer

Excitation640 nm laser
Emission660/20 nm filter
Instrument specification(s)APC channel

Fluorescence microscope

ExcitationCy5 filter set
EmissionCy5 filter set
Recommended plateBlack wall/clear bottom
Instrument specification(s)Cy5 filter set

Example protocol


PREPARATION OF STOCK SOLUTION

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

iFluor™ 647-streptavidin conjugate stock solution (1 mg/mL):
Add 200 µL (Cat#16966) and 1 mL (Cat#16996) ddH2O to make stock solution. Note: Store at 2-6ºC and kept away from light. To extend the shelf-life of this product, add an equal volume of glycerol to make a final concentration of approximately 50% glycerol and store at -20ºC.

PREPARATION OF WORKING SOLUTION

iFluor™ 647-streptavidin conjugate working solution:
For IF, the suggested staining concentration is at 75-750 ng/mL. For FACS, the suggested concentration is at 100 ng-1 µg/million cells in 1 mL staining buffer. Note: For the best performance of each application, the optimal concentration of this reagent needs to be carefully determined. Note: The working solutions can be made in aqueous buffers.

SAMPLE EXPERIMENTAL PROTOCOL

  1. Block and treat the samples with biotinylated antibodies of interest as per the manufacturer's recommendations.

  2. Add iFluor™ 647-streptavidin conjugate working solution in the samples at appropriate concentration.

  3. Incubate at room temperature for 30 minutes to 1 hour. Note: Optimal time for incubation needs to be determined carefully.

  4. Remove the working solution and resuspend the cells in your choice of buffer.

  5. Take the image using the fluorescence microscope or record the intensity using flow cytometer.

Calculators


Common stock solution preparation

Table 1. Volume of Water needed to reconstitute specific mass of iFluor™ 647-streptavidin conjugate to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.

0.1 mg0.5 mg1 mg5 mg10 mg
1 mM1.923 µL9.615 µL19.231 µL96.154 µL192.308 µL
5 mM384.615 nL1.923 µL3.846 µL19.231 µL38.462 µL
10 mM192.308 nL961.538 nL1.923 µL9.615 µL19.231 µL

Molarity calculator

Enter any two values (mass, volume, concentration) to calculate the third.

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles
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Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Correction Factor (260 nm)0.03
Correction Factor (280 nm)0.03
Correction Factor (656 nm)0.0793
Extinction coefficient (cm -1 M -1)2500001
Excitation (nm)656
Emission (nm)670
Quantum yield0.251

Citations


View all 1 citations: Citation Explorer
Overexpression of MACC1 and the association with hepatocyte growth factor/c-Met in epithelial ovarian cancer
Authors: Li, Hongyu and Zhang, Hui and Zhao, Shujun and Shi, Yun and Yao, Junge and Zhang, Yanyan and Guo, Huanhuan and Liu, Xingsuo
Journal: Oncology letters (2015): 1989--1996

References


View all 47 references: Citation Explorer
A streptavidin paramagnetic-particle based competition assay for the evaluation of the optical selectivity of quadruplex nucleic acid fluorescent probes
Authors: Largy E, Hamon F, Teulade-Fichou MP.
Journal: Methods. (2012)
Biotin-4-fluorescein based fluorescence quenching assay for determination of biotin binding capacity of streptavidin conjugated quantum dots
Authors: Mittal R, Bruchez MP.
Journal: Bioconjug Chem (2011): 362
Iminobiotin binding induces large fluorescent enhancements in avidin and streptavidin fluorescent conjugates and exhibits diverging pH-dependent binding affinities
Authors: Raphael MP, Rappole CA, Kurihara LK, Christodoulides JA, Qadri SN, Byers JM.
Journal: J Fluoresc (2011): 647
Streptavidin-Binding Peptide (SBP)-tagged SMC2 allows single-step affinity fluorescence, blotting or purification of the condensin complex
Authors: Kim JH, Chang TM, Graham AN, Choo KH, Kalitsis P, Hudson DF.
Journal: BMC Biochem (2010): 50
Determination of 17beta-oestradiol by fluorescence immunoassay with streptavidin-conjugated quantum dots as label
Authors: Sun M, Du L, Gao S, Bao Y, Wang S.
Journal: Steroids (2010): 400
Multimodality nuclear and fluorescence tumor imaging in mice using a streptavidin nanoparticle
Authors: Liang M, Liu X, Cheng D, Liu G, Dou S, Wang Y, Rusckowski M, Hnatowich DJ.
Journal: Bioconjug Chem (2010): 1385
Site-dependent excited-state dynamics of a fluorescent probe bound to avidin and streptavidin
Authors: Furstenberg A, Kel O, Gradinaru J, Ward TR, Emery D, Bollot G, Mareda J, Vauthey E.
Journal: Chemphyschem (2009): 1517
Influence of streptavidin on the absorption and fluorescence properties of cyanine dyes
Authors: Luschtinetz F, Dosche C, Kumke MU.
Journal: Bioconjug Chem (2009): 576
Fluorescent nanoscale detection of biotin-streptavidin interaction using near-field scanning optical microscopy
Authors: Park HK, Gokarna A, Hulme JP, Park HG, Chung BH.
Journal: Nanotechnology (2008): 235103
Application of biotin-4-fluorescein in homogeneous fluorescence assays for avidin, streptavidin, and biotin or biotin derivatives
Authors: Ebner A, Marek M, Kaiser K, Kada G, Hahn CD, Lackner B, Gruber HJ.
Journal: Methods Mol Biol (2008): 73