tPA assay kit

Total mouse tPA antigen assay

Cat. Num. AAA480485

• Gene Names: PLAT; TPA; T-PA
• Synonyms: tPA; Total mouse tPA antigen assay; Mouse tPA Total Antigen 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. 10x Wash Buffer: Dilute to 1x by adding 450 mL of DI water to the 50 mL of 10x Wash Buffer; prior; to use.; 2. TBS Buffer (100 mL): 0.1 M Tris-HCL, 0.15 M NaCl, pH 7.4; 3. 3% BSA Blocking Buffer (20 mL): 3% BSA (w/v) in TBS Buffer; 4. Standard: Reconstitute as directed on the vial to obtain a 1000 ng/mL standard stock solution; and; vortex gently to mix. Prepare immediately prior to use.; 5. Primary Antibody: Reconstitute with 3% BSA Blocking Buffer as directed on the vial and; vortex; gently to mix. Prepare immediately prior to use.; 6. Secondary Antibody: Dilute with 3% BSA Blocking Buffer as directed on the vial and vortex; gently; to mix. Prepare immediately prior to use.
• Standard Preparation: Reconstitute the Standard as directed on the vial to give a 100 ng/mL Standard Stock Solution. Do not prepare the standards until you are ready to apply them to the plate.; Table 1: Preparation of Standard Curve; Standard; tPA; Concentration; (ng/mL); Blocking; Buffer; (muL); Transfer; Volume; (muL); Transfer; Source; Final; Volume; (muL); S9 50 950 50 Stock Vial 500; S8 25 500 500 S9 600; S7 10 600 400 S8 500; S6 5 500 500 S7 600; S5 2 600 400 S6 500; S4 1 500 500 S5 500; S3 0.5 500 500 S4 500; S2 0.25 500 500 S3 600; S1 0.1 600 400 S2 1,000; B0 0 500 --- --- 500
• Sample Collection, Storage and Preparation: Samples of plasma, serum, cell culture media or other biological fluids may be applied directly to the plate. The assay measures tPA antigen in the 0.1-50 ng/ml range. Samples giving tPA levels above 50 ng/ml should be diluted in plasma devoid of tPA or 3% BSA 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: The Mouse tPA total antigen assay is intended for the quantitative determination of total plasminogen activator antigen in biological fluids. Tissue plasminogen activator (tPA) is a serine protease that converts plasminogen to the active serine protease plasmin in the blood fibrinolytic system1,
2. It also plays an important role in the removal of incipient thrombi
3. tPA is widely used for the thrombolytic treatment of acute myocardial infarction3.

Principle of assay: Mouse tPA will bind to the capture antibody coated on the microtiter plate. Free and complexed enzyme will react with the capture antibody on the plate. A standard calibration curve is prepared using dilutions of tPA along with the samples to be measured. After appropriate washing steps, monoclonal anti-mouse tPA primary antibody binds to the captured enzyme. Excess antibody is washed away and bound monoclonal antibody is then reacted with the secondary antibody conjugated to horseradish peroxidase. TMB substrate is used for color development at 450 nm.

References

1. Quax, Paul H. A., et al.: (1990) J. Biol. Chem.; 265(26):15560-15563
2. Kristensen, Paul, et al.: (1985) J. Cell Biol.; (101):305-311
3. Camani, Chantal, et al.: (1994) J. Biol. Chem.; 269(8):5770-5775
4. Declerck, P. J., et al.: (1995) Thromb Haemostas.; 74(5):1305-1309
5. Nassar, T., et al.: (2003) Blood; 103(3):897-902
6. Sakurama, T., et al.: (1994) Stroke; 25:451-456
7. The National Institute of Neurological Disorders and Stroke rt-PA Stroke Study Group: (1995) N Engl J
Med.;
333(24):1581-1588
8. Yepes, M., et al.: (2003) J Clin Invest.; 112(10):1483-1485
9. Underwood, M. J., et al.: (1993) Cardiovasc. Res.; 27(12):2270-2273

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]