tPA assay kit

Active porcine tPA functional assay

Cat. Num. AAA480486

• Gene Names: PLAT; TPA; T-PA
• Synonyms: tPA; Active porcine tPA functional assay; Porcine tPA Activity ELISA; tPA assay kit

• Sequence Length: 19

• Procedural Notes: 1. Use aseptic technique when opening and dispensing reagents.; 2. This kit is designed to work properly as provided and instructed. Additions, deletions, or substitutions to the procedure or reagents are not recommended, as they may be detrimental to the assay.; 3. Exercise universal precautions during the performance or handling of this kit or any component contained therein.
• Reagent Preparation: 1. TBS Buffer: 0.10 M TRIS, 0.15 M NaCl, pH 7.4; 2. Blocking Buffer: 3% BSA in TBS Buffer.; 3. 10x Wash Buffer: Dilute the 50 mL of concentrate to 1x with 450 mL of DI water prior to use.; 4. Biotinylated PAI-1: Reconstitute with Blocking Buffer as directed on the vial and vortex gently to mix. Prepare immediately prior to use.; 5. Standard: Reconstitute with Blocking Buffer as directed on the vial and vortex gently to mix.; Prepare according to include Standard Dilution Table immediately prior to use.; 6. Primary Antibody: Reconstitute with Blocking Buffer as directed on the vial and vortex gently to mix. Prepare immediately prior to use.; 7. Secondary Antibody: Dilute with Blocking Buffer as directed on the vial and vortex gently to mix.; Prepare immediately prior to use.
• Standard Preparation: Reconstitute the Standard according to the attached dilution table. Do not prepare the Standards until you are ready to apply them to the plate.
• Sample Collection, Storage and Preparation: Samples of porcine plasma in citrate or EDTA may be assayed with this kit. Plasma in heparin is not recommended. It is important to ensure a platelet free preparation as platelets can release PAI-1, which in turn could potentially form a complex with active tPA. Serum and cell culture media at neutral pH may also be used. For best results, collect 9 volumes of blood in 1 volume of 0.1M acidified citrate. The low pH of the resulting plasma insures that PAI-1 is inhibited from quenching tPA activity5,; 6. Immediately after collection of blood, samples must be centrifuges at 2500Xg for 15 minutes. The plasma must be transferred to a clean plastic tube and stored on ice prior to analysis. The tPA activity samples are stable for up to 5 hours on ice, up to one month frozen at -20 degree C, or up to 5 months frozen at -70 degree C. This assay measures active tPA in the 0.02-10 ng/mL range. If the sample is thought to have high tPA levels, dilutions may be made in a similar biological fluid devoid of tPA or in Blocking Buffer.
• Preparation and Storage: 1. Store this kit and its components at 4 degree C until use.; 2. Reconstituted standards and primary may be stored at -70C for later use. DO NOT; freeze/thaw the; Standards and Primary Antibody more than once.

Related Product Information for tPA assay kit

Introduction: This porcine tPA activity assay is intended for the quantitative determination of active tissue plasminogen activator in porcine plasma and other biological fluids. Tissue plasminogen activator is a serine protease that catalyzes the activation of plasminogen to plasmin1. Clinical studies have indicated that high tPA levels may increase the risk for thrombosis2, whereas decreased levels may cause neuronal plasticity and degeneration3

Principle of assay: Functionally active tPA will form a covalent complex with the biotinylated porcine PAI-1 which is bound to the avidin on the plate. After appropriate washing steps, polyclonal anti-porcine tPA primary antibody binds to the captured tPA. Excess antibody is washed away and bound polyclonal antibody is then reacted with the secondary antibody conjugated to horseradish peroxidase. Following an additional washing step, TMB is used for color development at 450nm. The amount of color development is directly proportional to the concentration of active tPA in the sample.

References

1. Sallés, Fernando J., et al.: (2002) J. Neuroscience 22(6):2125-2134
2. Wiman, Björn, et al.: (1984) J. Biol. Chem. 259(6):3644-47
3. Hastings, Gregg A., et al.: (1997) J. Biol. Chem. 272(52):33062-33067
4. Yang, Y.H., et al.: (2001) J. Immunol 167(2):1047-52
5. Ranby, M., et al.: (1989) Thromb Haemost 62(3):917-22
6. Chmielewska, J., et al.: (1986) Clin. Chem. 32(3):482-5
7. Kruithof, E.K., et al.: (1987) Blood 69(2):460-466
8. Aspelin, T., et al.: (2005) J. Thromb. Haemost. 3(9):1947-1954
9. Adrian, K., et al.: (2006) Artif. Organs 30(12):922-928
10. Nyberg, A., et al.: (2003) Acta. Anaesthesiol. Scand. 47:1125-1131

NCBI and Uniprot Product Information

• NCBI GI #: 2285954
• NCBI GeneID: 5327
• UniProt Accession #: Q6LBF5; P00750
• Molecular Weight: 2,342 Da
• NCBI Official Full Name: tPA, partial
• NCBI Official Synonym Full Names: plasminogen activator, tissue
• NCBI Official Symbol: PLAT
• NCBI Official Synonym Symbols: TPA; T-PA
• NCBI Protein Information: tissue-type plasminogen activator; alteplase; plasminogen activator, tissue type; plasminogen/activator kringle; reteplase; t-plasminogen activator; tissue plasminogen activator (t-PA)
• UniProt Protein Name: TPA protein
• Protein Family: Taurine--pyruvate aminotransferase
• UniProt Gene Name: tPA
• UniProt Entry Name: Q6LBF5_HUMAN

NCBI Description

This gene encodes tissue-type plasminogen activator, a secreted serine protease which converts the proenzyme plasminogen to plasmin, a fibrinolytic enzyme. Tissue-type plasminogen activator is synthesized as a single chain which is cleaved by plasmin to a two chain disulfide linked protein. This enzyme plays a role in cell migration and tissue remodeling. Increased enzymatic activity causes hyperfibrinolysis, which manifests as excessive bleeding; decreased activity leads to hypofibrinolysis which can result in thrombosis or embolism. Alternative splicing of this gene results in multiple transcript variants encoding different isoforms. [provided by RefSeq, Jul 2008]