Plasminogen activator inhibitor-1(PAI-1), a member of the serine protease inhibitor(serpin) super family. PAI-1 is the principal inhibitor of tissue plasminogen activator and urokinase, the activators of plasminogen and hence fibrinolysis. PAI-1 is mainly produced by the endothelium but is also secreted by other tissue types, such as adipose tissue. Source: Recombinant protein corresponding to aa24-402 from human PAI-1, fused to His-tag at N-terminal expressed in E. coli. Molecular Weight: ~45kD (400aa) Biological Activity: Measured by its ability to inhibit uPA cleavage of the substrate Z-GGR-AMC, the IC50 value is <56nM. Activity Assay: 1. Prepare a recombinant PAI-1 with various concentrations (2000nM, 1000nM, 500nM, 250nM, 125nM) in assay buffer. (Assay buffer: 50mM Tris-HCl pH 8.0, 0.01% Tween 20) 2. Mix equal volumes of the PAI-1 protein and diluted 2ug/ml uPA 3. Incubate at room temperature for 15 minutes. 4. After dilute incubated reaction mixtures (PAI-1, uPA) by a 5 fold dilution in assay buffer, mix 50ul of 200uM substrate (Z-GGR-AMC) and diluted mixtures. 5. Read excitation wavelength 355nm and emission wavelength 460nm for 10 minutes. Amino Acid Sequence: MGSSHHHHHH SSGLVPRGSH MVHHPPSYVA HLASDFGVRV GMAPALRHLY KELMGPWNKD EISTTDAIFV QRDLKLVQGF LTRLVLVNAL YFNGQWKTPF PDSSTHRRLF HKSDGSTVSV LTNILSAQLI SHWKGNMTRL PRLLVLPKFS LETEVDLRKP AVIVSARMAP EEIIMDRPFL FVVRHNPTGT VLFMGQVMEP FQQVAQASKD RNVVFSPYGV ASVLAMLQLT TGGETQQQIQ AAMGFKIDDK MPHFFRLFRS TVKQVDFSEV ERARFIINDW VKTHTKGMIS NLLGKGAVDQ PMMAQTNKFN YTEFTTPDGH YYDILELPYH GDTLSMFIAA PYEKEVPLSA LENLGMTDMF RQFQADFTSL SDQEPLHVAQ ALQKVKIEVN ESGTVASSST Storage and Stability: May be stored at 4C for short-term only. Aliquot to avoid repeated freezing and thawing. Store at -20C. Aliquots are stable for 6 months after receipt at -20C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.
