ANNEXIN V

In the early stages of apoptosis, changes can be observed on the cell surface. Cells lose their phospholipid membrane asymmetry and expose phosphatidylserine (PS) on the exterior plasma membrane. Annexin V is a Ca2+ dependent, phospholipid-binding protein with high affinity for phosphatidylserine (PS) and is thus very useful for identifying apoptotic cells exposing PS on the cell surface to the extracellular exvironment.

Annexin V may be conjugated to biotin or fluorophores such as FITC and phycoerythrin (PE) or it may be tagged with EGFP (Enhanced Green Fluorescent Protein). Since Annexin V conjugates or tagged Annexins retain their high affinity for PS, they are ideally suited as highly sensitive probes to identify early apoptotic cells and to detect apoptosis much earlier than assays based on DNA fagmentation or loss of membrane integrity. The stained cells can be evaluated by flow cytometry or fluorescence microscopy using common filter sets and flow cytometer channels.

Applications:

• Screening for apoptotic cells
• Differentiating apoptotic and necrotic cells
• Annexin detection
• Molecular imaging and Nuclear Medicine 
• Flow Cytometry
• Fluorescence microscopy

Annexin V (Cat.No. ANV0010)

• Source: E. coli (Recombinant)
• Molecular weight: 36 KD
• Purity: >98% by SDS-PAGE
• Concentration: 1mg/ml in 20 mM phosphate buffer (pH 7.5)
• Storage: Store at –80º C and avoid freeze-thaw cycles

Instruction:

Annexin A5 is used as a probe in the annexin A5 affinity assay to detect cells, which have expressed phosphatidylserine on the cell surface, a feature found in apoptosis as well as other forms of cell death. Apoptosis is a form of programmed cell death, which is used by the body to remove unwanted, damaged or senescent cells from tissues. Removal of apoptotic cells is carried out via phagocytosis by white blood cells such as macrophages or dendritic cells.

Phagocytic white blood cells recognize apoptotic cells by their exposure of negatively charged phospholipids (phosphatidylserine) on the cell surface. In normal cells the negative phospholipids reside on the inner side of the cellular membrane and the outer surface of the membrane is occupied by phospholipids, which do not have a charge. After cells have made the decision to commit suicide the negatively charged phospholipids are transported to the outer cell surface by a hypothetical protein known as scramblase. Phagocytic white blood cells express a receptor, which can detect the negatively charged phospholipids. After detection the apoptotic cells are removed. Phagocytes rapidly remove healthy individual apoptotic cells. However in pathological processes the removal of apoptotic cells may be delayed or even absent. Dying cells in tissue can be detected with annexin A5. Labeling of annexin A5 with fluorescent or radioactive molecules makes it possible to detect binding of labeled annexin A5 to the cell surface of apoptotic cells. After binding to the phospholipid surface, annexin A5 assembles into a cluster known as a trimer. This trimer consists of three annexin V molecules, which are bound to each other via non-covalent protein-protein interactions. The formation of annexin A5 trimers results in the formation of a two-dimensional crystal lattice on the phospholipid membrane. This clustering of annexin A5 on the membrane greatly increases the intensity of annexin A5 when labeled with a fluorescent or radioactive probe.

Annexin A5 has been successively used to detect apoptotic cells in vitro (cells in a culture tube) and in vivo (in laboratory mice and in patients in the clinic). Pathological processes in which apoptosis occurs include inflammation, ischemia damage of the heart cause by myocardial infarction, apoptotic white blood cells present in an atherosclerotic plaque in blood vessels, transplanted organs in the donor patient which are rejected by the immune system or tumor cells which are exposed to cytostatic drugs during chemotherapy.

Remarks: This molecule was shown to have a very good feature for labelling with fluorescent and radioactive probes.

Reference:

Gillies GM et al. Microfluidic reactor for the radiosynthesis of PET radiotracers.
Appl Radiat Isot. 2006 Mar;64(3):325-32. Epub 2005 Nov 15.

Here we show the first application of a microfabricated reaction system to PET radiochemistry, we term "microfluidic PET". The short half-life of the positron emitting isotopes and the trace chemical quantities used in radiolabelling make PET radiochemistry amenable to miniaturisation. Microfluidic technologies are capable of controlling and transferring tiny quantities of liquids which allow chemical and biochemical assays to be integrated and carried out on a small scale. Such technologies provide distinct advantages over current methods of PET radiochemical synthesis. To demonstrate "proof of principle" we have investigated the radiohalogenation of small and large molecular weight molecules using the microfluidic device. These reactions involved the direct radioiodination of the apoptosis marker Annexin V using iodine-124, the indirect radioiodination of the anti-cancer drug doxorubicin from a tin-butyl precursor and the radiosynthesis of 2-[(18)F]FDG from a mannose triflate precursor and fluorine-18 and hence provide a test bed for microfluidic reactions. We demonstrate the rapid radioiodination of the protein Annexin V (40% radiochemical yield within 1 min) and the rapid radiofluorination of 2-[(18)F]FDG (60% radiochemical yield within 4s) using a polymer microreactor chip. Chromatographic analysis showed that the labelling efficiency of the unoptimised microfluidic chip is comparable to conventional PET radiolabelling reactions.

Annexin-V-FITC Kits (Cat.No.ANV0011)

 Features and Benefits:

Detects apoptosis earlier in the process than DNA-based assays such as TUNEL.

• Rapid labeling of cells. Cell staining takes only 10 minutes.
• No cell fixation or processing required, reducing the detection time and allowing the cells to be used for further study.
• Propidium iodide secondary dye is included with the kit to differentiate apoptotic cells from viable and necrotic cells.

Preparation Principle:

Annexin V-FITC kit allows fluorescent detection of annexin V bound to apoptotic cells and quantitative determination by flow cytometry. The AnnexinV-FITC kit uses annexin V conjugated with fluorescein isothiocyante (FITC) to label phosphatidylserine sites on the membrane surface. The kit includes propidium iodide (PI) to label the cellular DNA in necrotic cells where the cell membrane has been totally compromised. This combination allows the differentiation among early apoptotic cells (annexin V positive, PI negative), necrotic cells (annexin V positive, PI positive), and viable cells (annexin V negative, PI negative).

Large scale and/or OEM production are welcome. We ganratee the best price for you.

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