Granzyme A (PE)

Intended Use: The reagent is designed for flow cytometric applications intended to identify and quantitate cells possessing cytoplasmic forms of the protein recognized by the monoclonal. Granzyme A is a member of the granzyme family of the serine proteases found specifically in the cytotoxic granules of cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. Granzyme A is the most abundant protease in CTL and NK cells. It induces caspase-independent cell death when introduced into target cells by perforin.1 Human Granzyme A is synthesized as a precursor (262 residues) with a signal peptide (residues 1-26), a propeptide (residues 27-28) and a mature chain (residues 29-262).2 The pro-enzyme is expressed and purified. After being activated by lysyl endopeptidase, rhGranzyme A cleaves a thioester substrate described above. Principle of the Test: Fixed cells are permeabilized, allowing conjugated antibodies access to proteins within the cell. Cells are initially fixed in order to minimize leakage of proteins out of the cell. The conjugated antibody is allowed to penetrate and bind to its target within the cell. Following a final wash, the cells are analyzed on a flow cytometer. Flow cytometric analysis of PE conjugates will generate a signal, which can be detected using 488 nm wavelength laser excitation and monitoring emitted fluorescence with a detector, optimized to collect peak emissions at 565-605nm. Additional Reagents Required: Paraformaldehyde Fixative-Dissolve 4.0 g of paraformaldehyde in 100 ml of sterile PBS (10 mM phosphate buffered saline, pH 7.4) by heating the solution at 56 degrees C for about 1 hour. All solids must be fully dissolved prior to use. Store buffer at 2 degrees -8 degrees C, protected from light, for no longer than 2 weeks. SAP buffer-Prepare a sterile solution containing 0.1% (w/v) saponin, 0.05% (w/v) NaN3 in Hanks