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Annexin A5 (ANXA5) Protein

Details for Product No. ABIN412014, Supplier: Log in to see
Protein Name
Reactivity
40
15
10
7
3
3
3
3
1
1
Host
61
9
8
2
1
1
Application
Fluorescence Microscopy (FM), Flow Cytometry (FACS)
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Purpose The bright and photo-stable reagent for detecting early stages of apoptosis.
Sample Type Cell Culture
Characteristics 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.
Background During apoptosis, phosphatidylserine (PS) is translocated from the cytoplasmic face of the plasma membrane to the cell surface. Annexin V has a strong, Ca2+ -dependent affinity for PS and therefore serves as a probe for detecting apoptosis.
Research Area Cardiovascular, Atherosclerosis, Apoptosis/Necrosis, Coagulation
Pathways Apoptosis
Application Notes Detection method: Flow cytometry (Ex/ Em- 488 /530 nm) using FITC signal detector (usually FL1) and fluorescence microscopy
Protocol A. Incubation of cells with Annexin V-EGFP:
1. Induce apoptosis by desired methods.
2. Collect 1 x 10^5 cells by centrifugation.
3. Resuspend cells in 500 µL of 1X Annexin V Binding Buffer (ABIN412353).
4. Add 1 µL of Annexin V-EGFP.
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 cells 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, trypsinize and gently wash cells with serum-containing medium 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, and cover with a glass coverslip. For analyzing adherent cells, grow cells directly on a coverslip. Following incubation (A.5), invert coverslip on a glass slide and visualize cells. The cells can also be washed with 1X Annexin V Binding Buffer and fixed in 2 % formaldehyebefore visualization. (Cells must be incubated with Annexin V-EGFP before fixation because 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 and rhodamine. Cells that have bound Annexin V-EGFP will show bright green staining on 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).
Restrictions For Research Use only
Storage 4 °C
Expiry Date 12 months
Product cited in: Schilling, Machkovech, He et al.: "TLR4 activation under lipotoxic conditions leads to synergistic macrophage cell death through a TRIF-dependent pathway." in: Journal of immunology (Baltimore, Md. : 1950), Vol. 190, Issue 3, pp. 1285-96, 2013 (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).

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).

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).

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).

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).

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).

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).