Brilliant Violet 510™ Annexin V

Product Details

Verified Reactivity

Human, Mouse, Rat

Reported Reactivity

Other Species

Formulation

Phosphate-buffered solution, pH 7.2, containing 0.09% sodium azide and BSA (origin USA).

Preparation

The protein was purified by affinity chromatography and conjugated with Brilliant Violet 510™ under optimal conditions.

Concentration

Lot-specific (to obtain lot-specific concentration and expiration, please enter the lot number in our Certificate of Analysis online tool.)

Storage & Handling

The 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.

Brilliant Violet 510™ excites at 405 nm and emits at 510 nm. The bandpass filter 510/50 nm is recommended for detection, although filter optimization may be required depending on other fluorophores used. Be sure to verify that your cytometer configuration and software setup are appropriate for detecting this channel. Refer to your instrument manual or manufacturer for support. Brilliant Violet 510™ is a trademark of Sirigen Group Ltd.

Learn more about Brilliant Violet™.

This product is subject to proprietary rights of Sirigen Inc. and is made and sold under license from Sirigen Inc. The purchase of this product conveys to the buyer a non-transferable right to use the purchased product for research purposes only. This product may not be resold or incorporated in any manner into another product for resale. Any use for therapeutics or diagnostics is strictly prohibited. This product is covered by U.S. Patent(s), pending patent applications and foreign equivalents.

Excitation Laser

Violet Laser (405 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 fluorochrome conjugated Annexin V.
4. Stain with a viability dye, such as PI (Cat. No. 421301), 7-AAD (Cat. Nos. 420403 & 420404), or Helix NP dyes (Cat. Nos. 425301, 425303, & 425305), if desired.
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. *For more concentrated samples, add a minimum of 200 µl of Annexin V Binding Buffer in this step.
7. 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

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. 25:295. PubMed
5. Grujic M, et al. 2010. J. Immunol. 185:1730. PubMed

Product Citations

1. Dickson P, et al. 2020. Cell Chem Biol. 27:1383. PubMed
2. Seo W, et al. 2020. Nat Commun. 11:1562. PubMed

RRID

not an antibody (BioLegend Cat. No. 640937)

Antigen Details

Biology Area

Cell Biology, Neuroscience

Gene ID

308 View all products for this Gene ID

UniProt

View information about Annexin V on UniProt.org

Documentation

• Technical Data Sheet (pdf)
• Certificate of Analysis
• Material Safety Data Sheet

Reviews

Related Protocols

• Cell Surface Flow Cytometry Staining Protocol

Related Pages & Pathways

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.

Certificate of Analysis