Alexa Fluor® 647 Annexin V

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Other Names
Annexin A5
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A647_Annexin-V_kit_052318
Human T leukemia cell line Jurkat, treated (left) or non-treated (right) with BioLegend’s anti-human CD95 (EOS9.1) mAb (Cat. No. 305704) for 4 hours, then stained with Annexin V- Alexa Fluor® 647 and Helix NP Green (Cat. No. 425303 at 1.25 nM) in Annexin V Binding buffer for 15 minutes at at 37°C.
  • A647_Annexin-V_kit_052318
    Human T leukemia cell line Jurkat, treated (left) or non-treated (right) with BioLegend’s anti-human CD95 (EOS9.1) mAb (Cat. No. 305704) for 4 hours, then stained with Annexin V- Alexa Fluor® 647 and Helix NP Green (Cat. No. 425303 at 1.25 nM) in Annexin V Binding buffer for 15 minutes at at 37°C.
See Alexa Fluor® 647 spectral data
Cat # Size Price Quantity Avail. Save
640911 25 tests 68€
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640912 100 tests 160€
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640943 300 tests 316€
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Description

Annexin V (or Annexin A5) is a member of the annexin family of intracellular proteins that binds to phosphatidylserine (PS) in a calcium-dependent manner. PS is normally only found on the intracellular leaflet of the plasma membrane in healthy cells, but during early apoptosis, membrane asymmetry is lost and PS translocates to the external leaflet. Fluorochrome-labeled Annexin V can then be used to specifically target and identify apoptotic cells.Annexin V Binding Buffer (cat. no. 422201) is recommended for use with Annexin V staining.Annexin V binding alone cannot differentiate between apoptotic cells and necrotic. Therefore, we recommend using our Helix NP™ Blue (Cat. No. 425305), Helix NP™ Green (Cat. No. 425303) or Helix NP™ NIR (Cat. No. 425301). Early apoptotic cells will exclude 7-AAD and PI, while late stage apoptotic cells and necrotic cells will stain positively, due to the passage of these dyes into the nucleus where they bind to DNA.

Product Details
Technical Data Sheet (pdf)

Product Details

Reactivity
All mammalian species
Formulation
Phosphate-buffered solution, pH 7.2, containing 0.09% sodium azide and 0.2% (w/v) BSA (origin USA).
Preparation
The purified protein was conjugated with Alexa Fluor® 647 under optimal conditions.
Concentration
Lot-specific (please contact technical support for concentration and total µg amount, or use our Lookup tool if you have a lot number.)
Storage & Handling
The Annexin V solution should be stored undiluted between 2°C and 8°C, and protected from prolonged exposure to light. Do not freeze.
Application

FC - Quality tested

Recommended Usage

Each lot of this product is quality control tested by immunofluorescent staining with flow cytometric analysis. For flow cytometric staining, the suggested use of this reagent is 5 µL per 100,000 - million cells in a 100 µL volume of Annexin V Binding Buffer (Cat No. 422201). It is recommended that the reagent be titrated for optimal performance for each application.

* Alexa Fluor® 647 has a maximum emission of 668 nm when it is excited at 633nm / 635nm.


Alexa Fluor® and Pacific Blue™ are trademarks of Life Technologies Corporation.

View full statement regarding label licenses
Excitation Laser
Red Laser (633 nm)
Application Notes

Annexin V Staining
1. Wash cells twice with cold BioLegend cell staining buffer (Cat. No. 420201) and then resuspend cells in Annexin V Binding Buffer (Cat. No. 422201) at a concentration of 1x106 cells/mL.
2. Transfer 100 µL of cell suspension in 5 mL test tube.
3. Add 5 µL of Alexa Fluor® 647 Annexin V.
4. Add 10 µL of PI solution (Cat. No. 421301) or 7-AAD (Cat. No. 420403/420404).
5. Gently vortex the cells and incubate for 15 min at RT (25°C) in the dark.
6. Add 400 µL of Annexin V Binding Buffer (Cat. No. 422201) to each tube. Analyze by flow cytometry.


For a better experience detecting apoptosis, we now recommend Apotracker™. Cell staining with Apotracker™ is Calcium independent. Thus, no special buffers are required, and the protocol can be shortened for single-step co-staining with other reagents.

Application References
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  8. Liu F, et al. 2011. Cancer Res. 71:6807. PubMed
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Product Citations
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  12. Mendelson A, et al. 2019. JCI Insight. 4:e126982. PubMed
  13. Rashid R, et al. 2019. Sci Data. 0.474305556. PubMed
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  17. Lin PB, et al. 2019. Oncol Rep. 41:3377. PubMed
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  22. Zeevi D, et al. 2010. J Cell Sci. 123:3112. PubMed
  23. Jin D, et al. 2010. Cancer Res. 70:2245. PubMed
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  25. Zimmerman M, et al. 2010. PLoS One. 5:e14076. PubMed
  26. Wortmann A, et al. 2011. J Biol Chem. 286:42303. PubMed
  27. Liu F, et al. 2011. Cancer Res. 71:6807. PubMed
  28. Zammarchi F, et al. 2011. Proc Natl Acad Sci U S A. 108:17779. PubMed
  29. Ho J, et al. 2011. PLoS One. 6:e16815. PubMed
  30. Notake T, et al. 2012. J Immunol. 188:4838. PubMed
  31. Yang D, et al. 2012. J Immunol. 188:4441. PubMed
  32. Liu F, et al. 2012. J Biol Chem. 287:25530. PubMed
  33. Schmid M, et al. 2012. Immunobiology. 217:610. PubMed
  34. Topalov N, et al. 2012. Arterioscler Thromb Vasc Biol. 32:2475. PubMed
  35. Xu L, et al. 2012. J Immunol. 189:3347. PubMed
  36. Leib P 2012. MBio. 16:267. PubMed
  37. Abaeva A, et al. 2013. J Biol Chem. 288:29621. PubMed
  38. Ponzetta A, et al. 2013. J Immunol. 191:5684. PubMed
  39. Beggs K, et al. 2014. Toxicol Sci. 137:91. PubMed
  40. Yue D, et al. 2014. Exp Cell Res. 322:149. PubMed
  41. Schott J, et al. 2014. PLoS Genet. 10:1004368. PubMed
  42. Matsumura M 2014. Sci Rep. 4:4852. PubMed
  43. Schögler A, et al. 2015. Eur Respir J. 45:428. PubMed
  44. Zakharova N, et al. 2015. PLoS One. 10:116665. PubMed
  45. Ghalei H, et al. 2015. J Cell Biol. 208:745. PubMed
  46. Herz J, et al. 2015. J Exp Med. 212: 1153 - 1169. PubMed
  47. Camacho K, et al. 2015. J Control Release. 210: 198-207. PubMed
  48. Yue D, et al. 2015. Exp Cell Res. 336: 141-149. PubMed
  49. Park A, et al. 2016. PLoS One. 11: 0148998. PubMed
  50. Artemenko E, et al. 2016. Biochem J. 473: 435 - 448. PubMed
  51. Lopez J, et al. 2016. Nat Commun. 7:10538. PubMed
  52. Bao W, et al. 2016. Sci Rep. 6:22579. PubMed
  53. Schlicher L, et al. 2016. EMBO Rep. 17: 1485 - 1497. PubMed
  54. Coe G, et al. 2016. Sci Rep. 6:30816. PubMed
  55. Podoplelova N, et al. 2016. Blood. 128: 1745 - 1755. PubMed
  56. Rossnagl S, et al. 2016. PLoS Biol. 14: 1002562. PubMed
  57. Mair B, et al. 2016. PLoS Genet. 12: 1006279. PubMed
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RRID
not an antibody (BioLegend Cat. No. 640911)
not an antibody (BioLegend Cat. No. 640912)
not an antibody (BioLegend Cat. No. 640943)

Antigen Details

Biology Area
Apoptosis/Tumor Suppressors/Cell Death, Cell Biology, Neuroscience
Gene ID
308 View all products for this Gene ID
UniProt
View information about Annexin V on UniProt.org

Related FAQs

How is your Annexin made and what sequence does it cover?

It is made in E. coli, covering human aa Met1-Asp320.

How does pH and staining temperature affect Annexin V-Phosphatidylserine binding?

Annexin-Phosphatidylserine binding is lost below pH 5.2 and with prolonged incubation over a temperature of 42°C.

Why do I need to use Annexin V Binding Buffer?

Annexin V binding requires the presence of calcium in the solution.  So, we provide Annexin V Binding Buffer (cat # 422201), which is optimized for the best performance of Annexin V staining.

Can I use RPMI during Annexin V staining?

It is best to follow protocol as described on the product data sheet. Moreover, RPMI 1640 has a relatively high concentration of phosphate and low calcium ion concentration, which negatively impacts Annexin binding to its target phosphatidylserine (PS). Measurement of cell death by using Annexin V may also be significantly affected by time of incubation on ice, calcium concentration, and type of medium.

Can I freeze Annexin V conjugates?

It should not be frozen as it will lead to loss of biological activity due to dimerization.

Is Annexin V suitable for conjugation with the Maxpar® kit for CyTOF®?

Maxpar® Labeling kits require the protein to be partially reduced, so the metal chelate can be introduced through an SH group in the hinge region of the reduced antibody. Human Annexin V contains only one Cysteine which was reported to be chemically inactive. Thus, the Maxpar® labeling protocol would not work with Annexin V, unless a free –SH group can be introduced to Annexin V.  For more information regarding SH-mediated conjugation of Annexin V please consult published papers such as this one.

Go To Top Version: 5    Revision Date: 04-20-2020

For research use only. Not for diagnostic use. Not for resale. BioLegend will not be held responsible for patent infringement or other violations that may occur with the use of our products.

 

*These products may be covered by one or more Limited Use Label Licenses (see the BioLegend Catalog or our website, www.biolegend.com/ordering#license). BioLegend products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products, reverse engineer functionally similar materials, or to provide a service to third parties without written approval of BioLegend. By use of these products you accept the terms and conditions of all applicable Limited Use Label Licenses. Unless otherwise indicated, these products are for research use only and are not intended for human or animal diagnostic, therapeutic or commercial use.

 

BioLegend Inc., 8999 BioLegend Way, San Diego, CA 92121 www.biolegend.com
Toll-Free Phone: 1-877-Bio-Legend (246-5343) Phone: (858) 768-5800 Fax: (877) 455-9587

This data display is provided for general comparisons between formats.
Your actual data may vary due to variations in samples, target cells, instruments and their settings, staining conditions, and other factors.
If you need assistance with selecting the best format contact our expert technical support team.

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