Granzyme B, also known as CTLA-1, is a serine protease found in the cytotoxic granules of cytotoxic T lymphocytes and NK cells and in articular chondrocytes. Granzyme B enters target cells through perforin channels and induces caspase-dependent apoptosis. It is upregulated in rheumatoid arthritis and cleaves vitronectin, fibronectin, and laminin. Human Granzyme B is synthesized as a precursor with a signal peptide (aa 1-18), a two amino acid propeptide, and a mature chain (aa 21-247). Mature human Granzyme B shares 69% amino acid sequence identity with mouse Granzyme B.
Produced from a hybridoma resulting from the fusion of a mouse myeloma with B cells obtained from a mouse immunized with purified, NS0-derived, recombinant human Granzyme B (rhGranzyme B; aa 19-247; Accession # P10144).
Western Blot, Flow Cytometry, Immunohistochemistry, Direct ELISA
Western Blot: This antibody can be used at 1-2ug/mL with the appropriate secondary reagents to detect human Granzyme B. Using a colorimetric detection system, the detection limit for rhGranzyme B is approximately 5 ng/lane under non-reducing and reducing conditions. Chemiluminescent detection will increase sensitivity by 5 to 50 fold.
Flow Cytometry: This antibody was tested for flow cytometry using NK-92 cells. For intracellular staining to detect human Granzyme B, cells must first be fixed and permeabilized using 4% paraformaldehyde and 0.1% saponin in PBS. Dilute this antibody to 25ug/mL and add 10ul of the diluted solution to 1-5 x 10e5 cells in a total reaction volume not exceeding 200ul. Following a 30 minute incubation, cells should be washed with 0.1% saponin prior to addition of a secondary developing reagent. The binding of unlabeled monoclonal antibodies may be visualized by adding 10ul of a 25ug/mL solution of a secondary developing reagent such as goat anti-mouse IgG conjugated to a fluorochrome. Cells should be washed for a final time in 0.1% saponin prior to flow cytometric analysis.
Immunohistochemistry: This antibody was used at a concentration of 8-25ug/mL with appropriate secondary reagents to detect human Granzyme B in paraformaldehyde-fixed human PBMC. For chromogenic detection of labeling, the use of Cell and Tissue Staining Kits is recommended.
Direct ELISA: This antibody can be used at 0.5-1.0ug/mL with the appropriate secondary reagents to detect human Granzyme B. The detection limit for rhGranzyme B is approximately 5 ng/well.
Optimal dilutions to be determined by the researcher.
Storage and Stability:
Lyophilized powder may be stored at 4 degrees C for short-term only. Reconstitute to nominal volume by adding sterile 40-50% glycerol and store at -20 degrees C. Reconstituted product is stable for 12 months at -20 degrees C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.