Annexin A5 (ANXA5) protein (EGFP)

Name: Annexin A5 (ANXA5)

Synonyms: PP4, ANX5, ENX2, Anx5, R74653, LC5, MGC93655, ANXA5, MGC126971, anx, anx5, ANX V, anxa5, cb989, wu:fa98f06, wu:fj10f10, MGC89158

Host: Please enquire

Reactivity: Please enquire

Conjugate: EGFP

Application: Flow Cytometry (FACS), Fluorescence Microscopy (FM)

Catalog no.: ABIN412014

Additional Information

Description: The bright and photo-stable reagent for detecting early stages of apoptosis. During apoptosis, phosphatidylserine (PS) is translocated from the cytoplasmic face of the plasma membrane to the cell surface. Annexin V has a strong, Ca²ᶧ -dependent affinity for PS and therefore serves as a probe for detecting apoptosis. The annexin V-EGFP fusion is brighter and photo-stable, ideal for detecting apoptosis by fluorescence microscopy with a FITC filter or by flow cytometry.

Application Details

Protocol: A. Incubation of cells with Annexin V-EGFP 1. Induce apoptosis by desired method. 2. Collect 1-5 x 10 5 cells by centrifugation. 3. Resuspend cells in 500 µl of 1X Binding Buffer (Cat.# 1035-100). 4. Add 1 µl of Annexin V-EGFP and 1 µl of propidium iodide (PI, Cat.# 1056-1) 5. Incubate at room temperature for 5 min in the dark. Proceed to B or C below depending on method of analysis. B. Quantification by Flow Cytometry Analyze Annexin V-EGFP binding by flow cytometry (Ex = 488 nm, Em = 530 nm) using FITC signal detector (usually FL1) and PI staining by the phycoerythrin emission signal detector (usually FL2). For adherent cells, gently trypsinize and wash cells once with serum-containing media before incubation with Annexin V-EGFP (A.3-5). C. Detection by Fluorescence Microscopy 1. Place the cell suspension from Step A.5 on a glass slide. Cover the cells with a glass coverslip. For analyzing adherent cells, grow cells directly on a coverslip. Following incubation (A.5), invert coverslip on glass slide and visualize cells. The cells can also be washed and fixed in 2% formaldehyde before visualization.

Application Notes: Note: Cells must be incubated with Annexin V-EGFP before fixation since any cell membrane disruption can cause nonspecific binding of Annexin V to PS on the inner surface of the cell membrane. 2. Observe the cells under a fluorescence microscope using a dual filter set for FITC & rhodamine. Cells which have bound Annexin V-EGFP will show green staining in the plasma membrane. Cells which have lost membrane integrity will show red staining (PI) throughout the nucleus and a halo of green staining (EGFP) on the cell surface (plasma membrane).

Storage: +4°C

Expiry Date: 12 months

Restrictions: For Research Use only

Publications

Product: Ozoren, Kim, Burns et al.: "The caspase 9 inhibitor Z-LEHD-FMK protects human liver cells while permitting death of cancer cells exposed to tumor necrosis factor-related apoptosis-inducing ligand." in: Cancer research, Vol. 60, Issue 22, pp. 6259-65, 2000 (PubMed).

Campbell, Bhat-Nakshatri, Patel et al.: "Phosphatidylinositol 3-kinase/AKT-mediated activation of estrogen receptor alpha: a new model for anti-estrogen resistance." in: The Journal of biological chemistry, Vol. 276, Issue 13, pp. 9817-24, 2001 (PubMed).

Goldberg, Jin, Ichikawa et al.: "Global effects of anchorage on gene expression during mammary carcinoma cell growth reveal role of tumor necrosis factor-related apoptosis-inducing ligand in anoikis." in: Cancer research, Vol. 61, Issue 4, pp. 1334-7, 2001 (PubMed).

Zelphati, Wang, Kitada et al.: "Intracellular delivery of proteins with a new lipid-mediated delivery system." in: The Journal of biological chemistry, Vol. 276, Issue 37, pp. 35103-10, 2001 (PubMed).

Snow, Chen, Nepomuceno et al.: "Resistance to Fas-mediated apoptosis in EBV-infected B cell lymphomas is due to defects in the proximal Fas signaling pathway." in: Journal of immunology (Baltimore, Md. : 1950), Vol. 167, Issue 9, pp. 5404-11, 2001 (PubMed).

Kuhn, Hunsucker, Chen et al.: "Targeted inhibition of the immunoproteasome is a potent strategy against models of multiple myeloma that overcomes resistance to conventional drugs and nonspecific proteasome inhibitors." in: Blood, Vol. 113, Issue 19, pp. 4667-76, 2009 (PubMed).

Neniskyte, Neher, Brown: "Neuronal death induced by nanomolar amyloid β is mediated by primary phagocytosis of neurons by microglia." in: The Journal of biological chemistry, Vol. 286, Issue 46, pp. 39904-13, 2011 (PubMed).