Annexin V Protein

Catalog No. ABIN2688832

Product Details

Target: Annexin V (ANXA5) (Annexin A5 (ANXA5))

Protein Type: Recombinant

Origin: Chemical

Source: Escherichia coli (E. coli)

Application: Flow Cytometry (FACS), Blocking Reagent (BR)

Brand: BD Pharmingen™

Characteristics: Purified Annexin V is routinely tested by flow cytometric analysis. Other applications were tested during antibody development only or reported in the literature. Flow cytometric analysis of Annexin V staining and blocking. Jurkat T cells were induced to undergo apoptosis by treatment with anti-Fas antibody, clone DX2 (Cat. No. 555670) and Protein G for three hours (first & second panels). Other cultures were left untreated (third & fourth panels). Cells were incubated with FITC Annexin V (first & third panels) or with recombinant Annexin V to block Annexin V binding sites, and then with FITC Annexin V (second & fourth panels). After a three hour treatment with Fas mAb, a population of cells was FITC Annexin V positive (first panel, M2 gate). FITC Annexin V staining was blocked when cells were first incubated with purified recombinant Annexin V (second panel). As expected, the cell population that was not treated with Fas mAb were primarily Annexin V negative (third panel). The small number of Annexin V positive cells in the untreated population likely represents a basal level of apoptosis. This was blocked when cells were first incubated with purified recombinant Annexin V (fourth panel). The M1 and M2 gates demarcate FITC Annexin V negative and positive populations, respectively. The addition of Protein G enhances the ability of DX2 to induce apoptosis, presumably by crosslinking the Fas receptor.

Recombinant Annexin V was expressed in bacteria and is ≥95% pure as determined by SDS/PAGE stained with Coomassie Blue. Analysis of purified recombinant Annexin V (1 μg) by SDS/PAGE. Gel was stained with Commasie Blue.

Purification: Purified

Application Details

Application Notes: Optimal working dilution should be determined by the investigator.

Comment:

BD Pharmingen™ Purified Recombinant Annexin V - Purified

Restrictions: For Research Use only

Handling

Concentration: 0.5 mg/mL

Buffer: Aqueous buffered solution containing ≤0.09 % sodium azide.

Preservative: Sodium azide

Precaution of Use: This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.

Handling Advice: Apoptosis is a normal physiologic process which occurs during embryonic development as well as in maintenence of tissue homeostasis. The apoptotic program is characterized by certain morphologic features, including loss of plasma membrane asymmetry and attachment, condensation of the cytoplasm and nucleus, and internucleosomal cleavage of DNA. Loss of plasma membrane is one of the earliest features. In apoptotic cells, the membrane phospholipid phosphatidylserine (PS) is translocated from the inner to the outer leaflet of the plasma membrane, thereby exposing PS to the external cellular environment. Annexin V is a 35-36 kDa Ca2+ dependent phospholipid-binding protein that has a high affinity for PS, and binds to cells with exposed PS. Annexin V may be conjugated to fluorochromes, such as fluorescein isothiocyanate (FITC), to serve as a sensitive probe for flow cytometric analysis of cells that are undergoing apoptosis. Since externalization of PS occurs in the earlier stages of apoptosis, Annexin V staining can identify apoptosis at an earlier stage than assays based on nuclear changes such as DNA fragmentation. In addition, Annexin V binding sites may be blocked by incubating cells with purified recombinant Annexin V prior to incubation with one of the fluorochrome labeled formats of Annexin V. Annexin V staining precedes the loss of membrane integrity which accompanies the latest stages of cell death resulting from either apoptotic or necrotic processes. Therefore, staining with Annexin V is typically used in conjunction with a vital dye such as 7-Amino-Actinomycin (7-AAD) to allow the investigator to identify early apoptotic cells (7-AAD negative, Annexin V positive). Viable cells with intact membranes exclude 7-AAD, whereas the membranes of dead and damaged cells are permeable to 7-AAD. For example, cells that are considered viable are both Annexin V and 7-AAD negative while cells that are in early apoptosis are Annexin V positive and 7-AAD negative, while cells that are in late apoptosis or already dead are both Annexin V and 7-AAD positive. This assay does not distinguish between cells that have undergone apoptotic death versus those that have died as a result of a necrotic pathway because in either case, the dead cells will stain with both Annexin V and 7-AAD. However, when apoptosis is measured over time, cells can be often tracked from Annexin V and 7-AAD negative (viable, or no measurable apoptosis), to Annexin V positive and 7-AAD negative (early apoptosis, membrane integrity is present) and finally to Annexin V and 7-AAD positive (end stage apoptosis and death). The movement of cells through these three stages suggests apoptosis. In contrast, a single observation indicating that cells are both Annexin V and 7-AAD positive, in of itself, reveals less information about the process by which the cells underwent their demise.

Storage: -80 °C

Storage Comment: Store product at -80°C prior to use or for long term storage of stock solutions. Avoid multiple freeze-thaws of product.

References

van Engeland, Ramaekers, Schutte, Reutelingsperger: "A novel assay to measure loss of plasma membrane asymmetry during apoptosis of adherent cells in culture." in: Cytometry, Vol. 24, Issue 2, pp. 131-9, (1996) (PubMed).

Homburg, de Haas, von dem Borne, Verhoeven, Reutelingsperger, Roos: "Human neutrophils lose their surface Fc gamma RIII and acquire Annexin V binding sites during apoptosis in vitro." in: Blood, Vol. 85, Issue 2, pp. 532-40, (1995) (PubMed).

Raynal, Pollard: "Annexins: the problem of assessing the biological role for a gene family of multifunctional calcium- and phospholipid-binding proteins." in: Biochimica et biophysica acta, Vol. 1197, Issue 1, pp. 63-93, (1994) (PubMed).

Koopman, Reutelingsperger, Kuijten, Keehnen, Pals, van Oers: "Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis." in: Blood, Vol. 84, Issue 5, pp. 1415-20, (1994) (PubMed).

Andree, Reutelingsperger, Hauptmann, Hemker, Hermens, Willems: "Binding of vascular anticoagulant alpha (VAC alpha) to planar phospholipid bilayers." in: The Journal of biological chemistry, Vol. 265, Issue 9, pp. 4923-8, (1990) (PubMed).

Target Details

Target: Annexin V (ANXA5) (Annexin A5 (ANXA5))

Alternative Name: Annexin V (ANXA5 Products)

Synonyms: anx Protein, anx5 Protein, ANX V Protein, anxa5 Protein, cb989 Protein, wu:fa98f06 Protein, wu:fj10f10 Protein, MGC89158 Protein, ANX5 Protein, ENX2 Protein, PP4 Protein, RPRGL3 Protein, Anx5 Protein, R74653 Protein, LC5 Protein, enx2 Protein, annexin A5 Protein, annexin A5b Protein, Annexin A5 Protein, annexin A5 L homeolog Protein, ANXA5 Protein, anxa5b Protein, anxa5 Protein, Anxa5 Protein, anxa5.L Protein

Background: Apoptosis is a normal physiologic process which occurs during embryonic development as well as in maintenence of tissue homeostasis. The apoptotic program is characterized by certain morphologic features, including loss of plasma membrane asymmetry and attachment, condensation of the cytoplasm and nucleus, and internucleosomal cleavage of DNA. Loss of plasma membrane is one of the earliest features. In apoptotic cells, the membrane phospholipid phosphatidylserine (PS) is translocated from the inner to the outer leaflet of the plasma membrane, thereby exposing PS to the external cellular environment. Annexin V is a 35-36 kDa Ca2+ dependent phospholipid-binding protein that has a high affinity for PS, and binds to cells with exposed PS. Annexin V may be conjugated to fluorochromes, such as fluorescein isothiocyanate (FITC), to serve as a sensitive probe for flow cytometric analysis of cells that are undergoing apoptosis. Since externalization of PS occurs in the earlier stages of apoptosis, Annexin V staining can identify apoptosis at an earlier stage than assays based on nuclear changes such as DNA fragmentation. In addition, Annexin V binding sites may be blocked by incubating cells with purified recombinant Annexin V prior to incubation with one of the fluorochrome labeled formats of Annexin V. Annexin V staining precedes the loss of membrane integrity which accompanies the latest stages of cell death resulting from either apoptotic or necrotic processes. Therefore, staining with Annexin V is typically used in conjunction with a vital dye such as 7-Amino-Actinomycin (7-AAD) to allow the investigator to identify early apoptotic cells (7-AAD negative, Annexin V positive). Viable cells with intact membranes exclude 7-AAD, whereas the membranes of dead and damaged cells are permeable to 7-AAD. For example, cells that are considered viable are both Annexin V and 7-AAD negative while cells that are in early apoptosis are Annexin V positive and 7-AAD negative, while cells that are in late apoptosis or already dead are both Annexin V and 7-AAD positive. This assay does not distinguish between cells that have undergone apoptotic death versus those that have died as a result of a necrotic pathway because in either case, the dead cells will stain with both Annexin V and 7-AAD. However, when apoptosis is measured over time, cells can be often tracked from Annexin V and 7-AAD negative (viable, or no measurable apoptosis), to Annexin V positive and 7-AAD negative (early apoptosis, membrane integrity is present) and finally to Annexin V and 7-AAD positive (end stage apoptosis and death). The movement of cells through these three stages suggests apoptosis. In contrast, a single observation indicating that cells are both Annexin V and 7-AAD positive, in of itself, reveals less information about the process by which the cells underwent their demise.

Pathways: Apoptosis