uPA assay kit

Active mouse uPA functional assay kit

Cat. Num. AAA480470

• Synonyms: uPA; Active mouse uPA functional assay kit; Mouse uPA Activity Assay; uPA assay kit

• Procedural Notes: 1. This assay should be performed at room temperature.; 2. If not using the entire plate at once, prepare only the appropriate amount of primary antibody, biotinylated PAI-1 and uPA standard. The remaining stock solutions should then be frozen and stored at -70 degree C. Viable for up to two weeks when stored at -70 degree C. All other components should remain refrigerated.; 3. Use aseptic technique when opening and removing reagents from vials and bottles.; 4. To minimize error due to handling, wipe the exterior bottom of the microplate wells with a lintfree paper towel.
• Reagent Preparation: The following solutions should be prepared fresh before starting the assay.; 1. TBS Buffer: 0.1 M TRIS, 0.15 M NaCl, pH 7.4; 2. 3% BSA 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 3% BSA Blocking Buffer as directed on the vial and vortex gently to mix. Prepare immediately prior to use.; 5. Standard: Reconstitute with 3% BSA Blocking Buffer as directed on the vial and vortex gently to mix. Prepare according to included Standard Dilution Table immediately prior to use.; 6. Primary Antibody: Reconstitute with 3% BSA Blocking Buffer as directed on the vial and vortex gently to mix. Prepare immediately prior to use.; 7. Secondary Antibody: Dilute with 3% BSA Blocking Buffer as directed on the vial and vortex gently to mix. Prepare immediately prior to use.
• Sample Collection, Storage and Preparation: Samples should be collected using 0.01 M trisodium citrate, acidified citrate or Stabilytetm collection media. Collection should be in accordance with the collection vial manufacturer's 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 for 15 minutes.; 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 24 hours when stored at 4 degree C with the Sabilytetm media or one month if stored at -20 degree C.; 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: Store the kit and all of its components at 4 degree C before use; 2. If not using the entire plate at once, prepare only the appropriate amount of Primary; Antibody,; Biotinylated PAI-1, and uPA Standard. The remaining stock solutions should be aliquoted,; frozen; and stored at At -70 degree C. DO NOT freeze/thaw the Primary Antibody or Standard more than; once.; All other components should remain refrigerated. Store unused portions of the microplate in; a; pouch with a desiccant at 4 degree C.

Related Product Information for uPA assay kit

Introduction: Urokinase-type Plasminogen Activator (uPA) is a glycosylated serine protease that is synthesized in endothelial and kidney epithelial cells. There are two forms of uPA, the low molecular weight (LMW ~31 kDa) and high molecular weight (HMW ~55 kDa). The HMW form undergoes autoproteolysis resulting in the LMW plus an 18.5 kDa amino terminal fragment (ATF). It is this ATF that has been shown to inhibit proliferation and invasion of cancer cells by binding to uPA receptors(1).

Principle of assay: This is an ELISA (Enzyme-Linked Immunosorbent Assay) for the quantitative analysis of active uPA levels in biological fluid. This test kit operates on the basis of sandwich ELISA where active uPA enzyme complexes with PAI-1 and is quantitated with the use of an HRP labeled secondary antibody. First the biotinylated PAI-1 binds to the avidin coated wells. Next, active uPA, present in the standard or unknown samples, complexes with PAI-1. Inactive or complexed uPA is removed in a subsequent wash step. A primary antibody specific for uPA 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. Declerck, P., et al., (1995) Thromb Haemostas., Novu 74(5):1305-1309
2. Singh, I., et al., (2003) Circulation 107(6):869-875
3. Kjøller, L., (2002) Biol. Chem. 383:5-19
4. Yang, Y., et al., (2001) J. Immunol. 167(2):1047-1052