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AAT Bioquest

iFluor® 647-streptavidin conjugate

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.

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.

Spectrum

Product family

NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (260 nm)Correction Factor (280 nm)
iFluor® 350-streptavidin conjugate3454502000010.9510.830.23
iFluor® 405-streptavidin conjugate4034273700010.9110.480.77
iFluor® 488-streptavidin conjugate4915167500010.910.210.11
iFluor® 514-streptavidin conjugate5115277500010.8310.2650.116
iFluor® 532-streptavidin conjugate5375609000010.6810.260.16
iFluor® 555-streptavidin conjugate55757010000010.6410.230.14
iFluor® 594-streptavidin conjugate58760320000010.5310.050.04
iFluor® 633-streptavidin conjugate64065425000010.2910.0620.044
iFluor® 680-streptavidin conjugate68470122000010.2310.0970.094
iFluor® 700-streptavidin conjugate69071322000010.2310.090.04
iFluor® 750-streptavidin conjugate75777927500010.1210.0440.039
iFluor® 546-streptavidin conjugate54155710000010.6710.250.15
iFluor® 568-streptavidin conjugate56858710000010.5710.340.15
iFluor® 790-streptavidin conjugate78781225000010.1310.10.09
iFluor® 800-streptavidin conjugate80182025000010.1110.030.08
iFluor® 820-streptavidin conjugate82285025000010.110.16
iFluor® 840-streptavidin conjugate8368792000001-0.20.09
iFluor® 860-streptavidin conjugate85387825000010.10.14
Phalloidin-iFluor® 647 Conjugate65667025000010.2510.030.03
Cholyl-iFluor® 647 conjugate65667025000010.2510.030.03
Show More (11)

Citations

View all 2 citations: Citation Explorer
Rational Strategy for Designing Peptidomimetic Small Molecules Based on Cyclic Peptides Targeting Protein--Protein Interaction between CTLA-4 and B7-1
Authors: Tsuihiji, Kumiko and Honda, Eiji and Kojoh, Kanehisa and Katoh, Shizue and Taguri, Tomonori and Yoshimori, Atsushi and Takashima, Hajime
Journal: Pharmaceuticals (2022): 1506
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
Page updated on November 23, 2024

Ordering information

Price
Unit size
200 ug
1 mg
Catalog Number
1696616996
Quantity
Add to cart

Additional ordering information

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Physical properties

Molecular weight

~52000

Solvent

Water

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 yield

0.251

Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22

Storage

Freeze (< -15 °C); Minimize light exposure
UNSPSC12352200

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
HeLa cells were incubated with mouse anti-tubulin and biotin goat anti-mouse IgG followed by AAT&rsquo;s iFluor®&nbsp;647-streptavidin conjugate (Red, Left) or streptavidin conjugated with Alexa Fluor<sup>&reg;</sup>&nbsp;647 (Red, Right), respectively.
HeLa cells were incubated with mouse anti-tubulin and biotin goat anti-mouse IgG followed by AAT&rsquo;s iFluor®&nbsp;647-streptavidin conjugate (Red, Left) or streptavidin conjugated with Alexa Fluor<sup>&reg;</sup>&nbsp;647 (Red, Right), respectively.
HeLa cells were incubated with mouse anti-tubulin and biotin goat anti-mouse IgG followed by AAT&rsquo;s iFluor®&nbsp;647-streptavidin conjugate (Red, Left) or streptavidin conjugated with Alexa Fluor<sup>&reg;</sup>&nbsp;647 (Red, Right), respectively.