iFluor™ 647-streptavidin conjugate

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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.
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Unit Size: Cat No: Price (USD): Qty:
16966 $75


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Additional Ordering Information
Telephone: 1-800-990-8053
Fax: 1-408-733-1304
Email: sales@aatbio.com
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Overview

Ex/Em (nm)654/669
SolventWater
Storage Freeze (<-15 °C)
Minimize light exposure
Category Immunology
Streptavidin Conjugates
Related
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.




Spectrum Advanced Spectrum Viewer

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Protocol


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This protocol only provides a guideline, and should be modified according to your specific needs.
Key parameters
Instrument:Fluorescence microscope
Excitation:Cy5 filter set
Emission:Cy5 filter set
Recommended plate:Black wall/clear bottom
Instrument specification(s):Cy5 filter set
  
Instrument:Flow cytometer
Excitation:APC channel
Emission:APC channel
Instrument specification(s):APC channel
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.
Example data analysis and figures

Figure 1. 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® 647 (Red, Right), respectively.
Disclaimer
AAT Bioquest provides high-quality reagents and materials for research use only. For proper handling of potentially hazardous chemicals, please consult the Safety Data Sheet (SDS) provided for the product. Chemical analysis and/or reverse engineering of any kit or its components is strictly prohibited without written permission from AAT Bioquest. Please call 408-733-1055 or email info@aatbio.com if you have any questions.





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References

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






Resources
 
Safety Data Sheet (SDS)


Documents
1. Fluorescent Labeling Probes & Kits

Certificate of Analysis