BackgroundAnnexin A5 (or annexin V) is a cellular protein of the annexin family. Annexin A5 has been proposed to play a role in the inhibition of blood coagulation by competing for phosphatidylserine binding sites with prothrombin, and also to inhibit the activity of phospholipase A1. Annexin A5 forms a shield around negatively-charged phospholipid molecules, which blocks the entry of phospholipids into coagulation (clotting) reactions. Antibodies directed anti annexin A5 are the cause of the antiphospholipid syndrom in which the formation of the shield is disrupted by antibodies. Without the shield, there is an increased quantity of phospholipid molecules on cell membranes, speeding up coagulation reactions and causing the blood-clotting characteristic of the antiphospholipid antibody syndrome. Annexin A5 is used as a probe in the annexin A5 affinity assay to detect cells that have expressed phosphatidylserine on the cell surface, a feature found in apoptosis aswell as other forms of cell death (1,2). Platelets also expose phosphatidyl-serine on their surface when activated, which serves as binding site for various coagulation factors. Annexin A5 has been shown to interact with kinase insert domain receptor (3) and integrin beta 5 (4). RUU-WAC2a is a monoclonal that specifically reacts with annexin A5. This monoclonal antibody is directed to the phospholipid binding domain of annexin A5 and as such it inhibits annexin A5 binding to anionic phospholipid membranes for more than 95%. RUU-WAC 2A does not remove annexin A5 bound to phospholipid surfaces indiCating that the epitope is not accessible after interaction with annexin A5.

SourceRUU-WAC2a is a Mouse monoclonal IgG1 antibody obtained by fusion of P3-X63-Ag 8,653 Mouse myeloma cells with spleen cells from a BABL/c Mouse immunized with recombinant annexin A5.

ProductEach vial contains 100 ul of 1 mg/ml purified monoclonal antibody in PBS containing 0.09% sodium azide.

Formulation: Each vial contains 100 ul of 1 mg/ml purified monoclonal antibody in PBS containing 0.09% sodium azide.

SpecificityRUU-WAC2A reacts anti Human recombinant and enDogenous annexin A5. It shows no cross reactivity with Annexins I, II, III, IV, VI, VII and VIII in ELISA or in immunoprecipitation. It does not recognise Monkey and Rat Annexin V in ELISA.

ApplicationsThe monoclonal antibody is suitable for ELISA in combination with a polyclonal antibody raised anti Annexin V in Rabbit. It can be applied in immunofluorescence to parafolmaldehyde fixed cell samples and in immunohistochemistry of frozen sections and formaldehyde fixed and paraffin embedded tissue specimens. For immunohistochemistry of paraffin sections a pretreatment step with 2% amino-propyl-triethoxysilan (in 100% ethanol) and 0,2 % BSA is recommended. The sections are heated for 15 minutes at 100°C in a citRate buffer (10 mM trisodium citRate, pH 6,0). The sections are then further processed by routine procedures (see below). RUU-WAC2a inhibits annexin A5 binding to anionic phospholipid-containing vesicles, activated platelets and apoptotic cells for more than 95%. Optimal antibody dilution should be determined by titration; recommended range is 1:50 – 1:100 for immunohistochemistry with avidin-biotinylated Horseradish peroxidase complex (ABC) as detection reagent, and 1:100 – 1:500 for immunoblotting applications.

StorageThe antibody is shipped at ambient temperature and may be stored at +4°C. For prolonged storage prepare appropriate aliquots and store at or below -20°C. Prior to use, an aliquot is thawed slowly in the dark at ambient temperature, spun down again and used to prepare working dilutions by adding sterile phosphate buffered saline (PBS, pH 7.2). Repeated thawing and freezing should be avoided. Working dilutions should be stored at +4°C, not refrozen, and preferably used the same day. If a slight precipitation occurs upon storage, this should be removed by centrifugation. It will not affect the performance or the concentration of the product.

CautionThis product is intended FOR RESEARCH USE ONLY, and FOR TESTS IN VITRO, not for use in diagnostic or therapeutic procedures involving humans or animals. It may contain hazardous ingredients. Please refer to the Safety Data Sheets (SDS) for additional information and proper handling procedures. Dispose product remainders according to local regulations.This datasheet is as accurate as reasonably achievable, but Exalpha Biologicals accepts no liability for any inaccuracies or omissions in this information.

References1. Koopman, G., Reutelingsperger, C.P., Kuijten, G.A.M. et al. (1994). Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 84, 1415-20. 2. Vermes, I., Haanen, C., Steffens-Nakken, H. and Reutelingsperger, C. (1995). A novel assay for apoptosis-flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J Immunol Methods 184, 39. 3. Wen, Y., Edelman J.L., Kang, T. and Sachs, G. (1999). Lipocortin V may function as a signaling protein for vascular endothelial growth factor receptor-2/Flk-1. Biochem. Biophys. Res. Commun. 258, 713–21. 4. Cardó-Vila, M., Arap, W. and Pasqualini, R. (2003). Alpha v beta 5 integrin-dependent programmed cell death triggered by a peptide mimic of annex V. Mol. Cell 31, 1151-62. 5. Van Heerde, W.L. (1994). Thesis; localization, plasma levels and anticoagulant activity. 6. Van Heerde, W.L., Reutelingsperger, C.P., Maassen, C., Lux, P., Derksen, R.H. and De Groot, P.G. (2003). The presence of antiphospholipid antibodies is not related to increased levels of annexin A5 in plasma. Jn Thromb Haemost. 3, 532-536. 7. Van Heerde, W.L., Lap, P., Schoormans, S., De Groot, P.G., Reutelingsperger, C.P.M. and Vroom, T.M. (2004). Localization of annexin A5 in Human tissues. Annexins 1, 37-43.

Protein Reference(s)

Database Name: UniProt

Accession number: P08758

Safety Datasheet(s) for this product:EA_Sodium Azide/wp-content/uploads/SDS/Antibody SDS with Sodium AzideV2.pdf