Urokinase activity is measured by
cleaving a synthetic 7-amino-4-trifluoromethylcoumarin
(AFC)-based substrate to yield AFC, a fluorescent
product (λex = 350/ λem = 450 nm), proportional to the
enzymatic activity
minimize adherence and/or clumping of the Mφ.
14. Centrifuge the peritoneal cells for 5 minutes at
350 X g, at 4 °C. The cells can then be re-
suspended and washed in saline or immuno-
fluorescence wash
/ml
Denaturing gel9
Prepare a 20% denaturing gel containing 7 M urea and
pre-run the gel for 30-60 min.
Cell Lysate. Use 70-350 µg protein of HeLa cell lysate
per test. (For other cell lysates
C) preferably in the dark, after addition of
enzyme conjugate to the last well.
7. Wash the wells 6 times with 350 µL of distilled
water using either a suitable plate washer or wash
bottle taking