uPA assay kit

Total Mouse uPA functional assay kit

Cat. Num. AAA480471

• Synonyms: uPA; Total Mouse uPA functional assay kit; Total Mouse uPA Assay; uPA assay kit

• Procedural Notes: 1. Always use new pipette tips for the buffer, conjugate, standards, samples etc.; 2. Before pipetting a reagent, rinse the pipette tip three times with that reagent (i.e. fill the tip with the desired amount of reagent and dispense back into the same vial - repeat 2 times). Now the tip is properly rinsed and ready to dispense the reagent into your well or test tube.; 3. When pipetting into the wells, DO NOT allow the pipette tip to touch the inside of the well, or any of the reagents already in the well - this can cause cross contamination.; 4. Standards and samples should be assayed in duplicate.; 5. To quantitate, always run a standard curve when testing samples.; 6. Gently mix specimens and reagents before use. Avoid vigorous agitation.; 7. Before taking an absorbance reading, wipe the outside bottom of the wells with a lintfree wiper to remove dust and fingerprints.; 8. 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. Remove excess air before sealing.; 9. If not using the entire plate at once, prepare only the appropriate amount of primary antibody and uPA standard. The remaining stock solutions should then be frozen andstored at -70 C. Viable for up to two weeks when stored at -70 C. All other components should remain refrigerated.
• Reagent Preparation: The following solutions should be prepared fresh before starting the assay.; -TBS Buffer: 0.10 M TRIS, 0.15 M NaCl, pH 7.4; -Blocking Buffer: 3% BSA in TBS buffer (indicated above or as provided)
• Sample 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 24 hours when stored at 4 degree C with the Sabilytetm media.; If you expect high concentrations of uPA from your samples they can be diluted with 3%; BSA blocking buffer (see reagent preparation below).; 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: 4 degree C

Related Product Information for uPA assay kit

Description: Urokinase-type Plasminogen Actvator (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 and 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 total uPA levels in biological fluid. This test kit operates on the basis of sandwich ELISA where active, latent and complexed 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, latent or complexed uPA present in the standard or unknown, complexes with PAI-1. A primary antibody specific for the various uPA forms is then added to each well followed by the HRP conjugated secondary antibody. The 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. Luparello C et al. (1996) Eu J Cancer A: 702-707