Lacritin PKCa Signaling Assay¶
Contributed by Gordon W. Laurie, School of Medicine, University of Virginia, United States
Lacritin PKCa Signaling Assay
- Seed Cells in serum-containing media overnight in 24-well plates at a density of 0.5 x 105 cells/mm2 corresponding to 20 – 30% confluency after cell attachment.
- Incubate cells in serum-free medium without supplements overnight to 24 hours.
- Reconstitute lacritin or lacritin fragment in water or serum-free medium to 10 µM stock. Wash cells with serum-free media without supplements three times and then replace media with serum-free medium containing lacritin (1 - 1000 nM), PMA (100 nM), EGF (1.6 nM) or 10% fetal bovine serum. In negative controls, incubate cells with equal molar amounts of C-25 or bovine serum albumin or without additive in serum-free media without supplements.
- Incubate for 1 – 15 min in the hood at room temperature.
- Stop incubation by adding ice-cold PBS containing sodium orthovanadate (2 mg/ml).
- Aspirate PBS and extract cells in 1% NP40 containing sodium orthovanadate (2 mg/ml), DTT (5 mg/ml), protease inhibitors (1 µg/ml leupeptin, 2 µg/ml aprotinin, 0.4 mg/ml sodium fluoride), 50 mM HEPES, 100 mM NaCl and 2 mM EDTA.
- Separate by 10% SDS PAGE, transfer and blot.
- Detect with anti-phospho-PKCa/bII (Cat. no. 9375) from Cell Signaling Technology (Beverly MA). or for loading control with anti-PKCa (Cat. no. 05-154) from Millipore-Upstate (Temecula CA). For other phospho signaling, detect with anti-PLCg2 (Cat. no. 3874) or antiphosphotyrosine antibody (P-Tyr-100; Cat. no. 9411) also from Cell Signaling Technology.
- After washing, bound antibodies were detected with peroxidase-labeled secondary antibody and ECL.
This method is based, with permission, on an original protocol available here.