Purified Annexin V

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Other Names
Annexin A5
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Annexin-V_Purified_FC_1_050714
Human T cell leukemia cell line, Jurkat, was treated (4 hours) with (top 2 panels) or without (bottom two panels) LEAF™ purified anti-CD95 (clone EOS9.1), then stained with Annexin V FITC (panels 1 & 3) or with purified recombinant Annexin V to block Annexin V FITC staining (panels 2 & 4).
  • Annexin-V_Purified_FC_1_050714
    Human T cell leukemia cell line, Jurkat, was treated (4 hours) with (top 2 panels) or without (bottom two panels) LEAF™ purified anti-CD95 (clone EOS9.1), then stained with Annexin V FITC (panels 1 & 3) or with purified recombinant Annexin V to block Annexin V FITC staining (panels 2 & 4).
  • Annexin-V_Purified_FC_2_050714
Cat # Size Price Quantity Avail. Save
640901 100 µg $200
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640902 500 µg $625
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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 and necrotic cells. 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

Product Details

Reactivity
All mammalian species
Formulation
Phosphate-buffered solution, pH 7.2, containing 0.09% sodium azide.
Preparation
The protein was purified by affinity chromatography.
Concentration
0.5 mg/ml
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 application, the suggested use of this reagent is 5 - 15 µg 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.

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 purified 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 room temperature (25°C), in the dark. 
6. Add 400 µl of Annexin V Binding Buffer (Cat. No. 422201) to each tube. Analyze by flow cytometry.

Application References

(PubMed link indicates BioLegend citation)
  1. Koopman G, et al. 1994. Blood 84:1415.
  2. Vermes I, et al. 1995. J. Immunol. Methods 184:39.
  3. Dachary-Prigent J, et al. 1993. Blood 81:2554.
  4. Sekine C, et al. 2009. Int Immunol. PubMed
  5. Grujic M, et al. 2010. J. Immunol. 185:1730. PubMed
Product Citations
  1. Abate M,et al. 2017. Sci Rep. . 10.1038/s41598-017-14495-6. PubMed
  2. Jacobs R, et al. 2017. J Immunol Methods. . 10.1016/j.jim.2017.09.001. PubMed
Publication Library
RRID
AB_2561295 (BioLegend Cat. No. 640901)
AB_2562279 (BioLegend Cat. No. 640902)

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

Can I freeze Annexin V conjugates?

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

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.

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.

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

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

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.

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.

Go To Top Version: 3    Revision Date: 11/03/2016

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., 9727 Pacific Heights Blvd, San Diego, CA 92121 www.biolegend.com
Toll-Free Phone: 1-877-Bio-Legend (246-5343) Phone: (858) 768-5800 Fax: (877) 455-9587

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