Detection of granzyme B in permeabilized human CD8+ T lymphocytes (filled profile). Isotype control in combination with a secondary FITC-labeled antibody is shown as control.Method: Permeabilized human T cells (5 x 105) were incubated on ice for 30 min in 50 µl FACS buffer (PBS, 5% Fetal calf serum, 0.02% azide) containing 1µg of FITC- labelled ab17929 antibody. After washing in FACS buffer, cells were anlyzed by flow cytometry.
Detection of granzyme B in a human CD8+ T cell clone.Method: Human T cells were plated onto polylysine treated glass slides, fixed and permeabilized in methanol at -20oC for 5 min, then in acetone at -20oC for 30 sec. After 3 washes in PBS, 0.1% BSA, slides were incubated for 1 hr at room temperature (RT) with 20 µg/ml of the non-conjugated form of ab17929 antibody in PBS, 0.1% BSA. After rinsing in PBS, a FITC-conjugated anti-mouse IgG was added for 30 min., slides washed again in PBS and visualized using a fluorescence microscope.
Detection of recombinant granzyme B. Granzyme B migrates as a 27 kDa species. Lane 1: 40 mg of lysate Lane 2: 4 mg of lysate Lane 3: 0.4 mg of lysate Method: Recombinant granzyme B was resolved by SDS-PAGE under reducing conditions, transferred to nitrocellulose and probed with the monoclonal anti-granzyme B non-conjugated form of antibody ab17929 at 1µg/ml. Proteins were visualized using a peroxidase-conjugated antibody to mouse IgG (SBA) and a chemiluminescence detection system.