Detection of femtomole quantities of mature cathepsin K with zymography
Section snippets
Cell culture
Human Thp-1 acute monocytic leukemia cells (American Type Culture Collection [ATCC]) were cultured in RPMI medium 1640 (Mediatech) containing 10% fetal bovine serum (FBS), 0.05% β-mercaptoethanol, 1% l-glutamine, and 1% penicillin/streptomycin. Cells were maintained with 5% CO2 at 37 °C. For macrophage differentiation, monocytes were incubated with 100 nM phorbol myristate acetate (PMA) for 24 h, followed by incubation for an additional 3 days in growth medium. RAW 264.7 murine macrophage cells
Cathepsin K activity detected by gelatin zymography is more sensitive than Western blot detection of mature cathepsin K
Procathepsin K was incubated in 0.1 M acetate buffer (pH 3.9), 10 mM DTT, and 5 mM EDTA for autocatalytic cleavage into the mature active form. Nonactivated procathepsin K (37.5 ng) and preactivated (cleaved) cathepsin K (37.5 ng) were then separately loaded for Western blots and cathepsin zymography (Fig. 1). The anti-cathepsin K antibody detects both the pro- and mature forms of cathepsin K, as seen in the 37-kDa bands of the nonactivated (0 min) sample and in the 29-kDa bands of mature cathepsin K
Discussion
Using recombinant cathepsin K and monocyte-derived macrophages, we have demonstrated a sensitive quantitative method of detecting femtomole amounts of mature cathepsin K activity by protecting the enzymes from irreversible denaturation with freshly added leupeptin and refolding them into active conformations after mild denaturation in 0.1% SDS. This simple electrophoresis method uses readily available and inexpensive reagents, and it visualizes the protein molecular mass to assert that the
Acknowledgments
Special thanks go to Randy Ankeny and Hannah Song for critical reading of this manuscript. Funding sources include the FACES/SURE program at Georgia Tech and Georgia Tech startup funds.
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