tPA assay kit

Rat tPA EIA Active ELISA kit

Cat. Num. AAA480459

• Gene Names: PLAT; TPA; T-PA
• Synonyms: tPA; Rat tPA EIA Active ELISA kit; Active Rat tPA Assay; tPA assay kit

• Sequence Length: 19

• Procedural Notes: 1. This assay should be performed at room temperature.; 2. Performance of the entire kit at once is not required. When performing this kit in part, please adhere to the following:; - All unused components should be returned to storage at 4 degree C.; - Desiccant bag must remain in foil pouch with unused strips. Keep pouch sealed when not in use to maintain a dry environment. Seal with a heat sealer. If a heat sealer is not available, thoroughly close the open end with tape. Try to remove excess air before sealing.; - If not using the entire plate at once, then prepare only the required amount of reagents where appropriate.; - tPA Standard and reconstituted biotinylated PAI-1 stock solutions must be stored at -70 degree C after initial use.; 3. To minimize error due to handling, wipe the exterior bottom of the microplate wells with a lint-free paper towel.
• Reagent Preparation: The following solutions should be prepared fresh before starting the assay.; 1. TBS Buffer: Dilute the 10x TBS Buffer to 1x with deionized water and mix prior to use.; 2. Blocking Buffer: 3% BSA in TBS Buffer.; 3. Wash Buffer: Dilute the 10x Wash Buffer to 1x with deionized water and mix prior to use.
• Sample Collection, Storage and Preparation: Samples should be collected using trisodium citrate, acidified citrate or Stabilitetm (DiaPharma) collection media. Collection should be in accordance with the collection vials manufacturers instructions or in a 1:10 ratio of collection media to blood.; Immediately, upon collection of blood, the samples should be centrifuged at 3000 x g. This should ensure the removal of platelets as they can release PAI-1 that in turn complexes with uPA. The plasma can be transferred to a clean plastic tube and stored frozen for up to one month. Samples are stable for approximately 5 hours when stored at 4 degree C with the Sabilytetm media or up to one month when stored at -20C.; Note: Detergents such as Triton X cause interference with the assay. If using detergent extracted samples, it is necessary to dialyze the samples overnight to remove the detergent.
• Preparation and Storage: 1. Store this kit and its components at 4 degree C until use.; 2. Do not freeze.

Testing Data

Testing Data #2

Related Product Information for tPA assay kit

Introduction: Tissue-type Plasminogen Activator (tPA) is a member of the serine proteinase family. tPA functions to lyse fibrin clots into soluble plasmin fragments. tPA is active in two forms, single-chain and two-chain. The two-chain tPA is created via interaction with the plasmin product cleaving the single chain. This two-chain form is regarded as the more active form. Both single-chain and two-chain tPA are complexable with PAI-1. PAI-1 acts as an inhibitor for tPA by binding to the tPA and thus stifling its ability to lyse fibrin. tPA can serve as an indicator of both myocardial infarction for patients with impaired fibrinolytic systems as well as a marker for type-II diabetes.

Principle of assay: This is an ELISA (Enzyme-Linked Immunosorbent Assay) for the quantitative analysis of active tPA levels in biological fluid. This test kit operates on the basis of sandwich ELISA where free, active, tPA enzyme complexes with PAI-1 and is quantified with the use of an HRP labeled secondary antibody. First the biotinylated PAI-1 binds to the avidin coated wells. Next, active tPA present in the standard or unknown, complexes with PAI-1. Inactive or complexed tPA is removed in a subsequent wash step. A primary antibody specific for tPA is then added to each well followed by the HRP conjugated secondary antibody. The bound conjugated secondary antibody is detected by the addition of substrate, which generates an optimal color after 10 minutes. Quantitative test results may be obtained by the measure and comparison of the sample and standard absorbance readings when read with a microplate reader at 450 nm.

References

1. Thogersen A et al. (1998) Circulation 98: 2241-2247
2. Eliasson M et al. (2003) Cardiovascular Diabetology 2:19

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]