PAI-1 Inhibition Kinetic Assay with tPA

PAI-1 Inhibition of tPA
1. tPA Control Rate – Incubate 5 ug of human or mouse tPA in a total volume of 100 ul TBS for 1 minute. Add 900 ul TBS. Add 2 ul Pefachrome tPA substrate at 10 mM (purchased from Centerchem), mix and take kinetic absorbance at 405 nm for 1 minute.
2. PAI-1 Inhibition Control Rate – Incubate 5 ug of tPA with 2 ug of fully active control PAI-1 in a total volume of 100 ul TBS for 1 minute. Add 900 ul TBS. Add 2 ul Pefachrome tPA substrate, mix and take kinetic absorbance at 405 nm for 1 minute.
If the PAI-1 is 100% active, it should inhibit about 65% of the active tPA. The rate should be decreased 65% compared to the tPA Control Rate.
3. PAI-1 Sample Rate – Incubate 5 ug of tPA with 2 ug of sample PAI-1 in a total volume of 100 ul TBS for 1 minute. Add 900 ul TBS. Add 2 ul Pefachrome tPA substrate, mix and take kinetic absorbance at 405 nm for 1 minute.
Compare the sample rate to the PAI-1 Inhibition Control Rate and the tPA Control Rate. If the PAI-1 in your sample has gone partly latent, it will inhibit less tPA than the 100% active PAI-1, so the rate will be greater than the PAI-1 Inhibition Control Rate. If the PAI-1 is completely latent, the rate will be equal to or greater than the tPA Control Rate. You can estimate the percent activity remaining in the sample by taking a ratio of the rates.

PAI-1 Inhibitory Antibody
Perform 1. tPA Control Rate and 2. PAI-1 Inhibition Control Rate as above.
3. Antibody Control Rate – Incubate 2 ug control PAI-1 with 3 ug of control inhibitory antibody in a total volume of 75 ul TBS for 5 minutes. Add 5 ug of tPA, increase the total reaction volume to 100 ul with TBS and incubate an additional 1 minute. Add 900 ul TBS. Add 2 ul Pefachrome tPA substrate, mix and take kinetic absorbance at 405 nm for 1 minute.
Compare this rate to the PAI-1 Inhibition Control Rate and the tPA Control Rate. The antibody should inhibit about 80% of the PAI-1, which will only inhibit approximately 13% of the tPA. The rate should be close to the tPA Control Rate.
4. Antibody Sample Rate – Incubate 2 ug control PAI-1 with 3 ug of sample inhibitory antibody in a total volume of 75 ul TBS for 5 minutes. Add 5 ug of tPA, increase the total reaction volume to 100 ul with TBS and incubate an additional 1 minute. Add 900 ul TBS. Add 2 ul Pefachrome tPA substrate, mix and take kinetic absorbance at 405 nm for 1 minute.
If the antibody has lost its inhibitory activity, the rate will be closer to the PAI-1 Inhibition Control Rate than the Antibody Control Rate.

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