Western blot analysis of extracts from CD56+ NK cells, NK-92 cells, and SU-DHL-1 cells using Granzyme H (E4T2E) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected (+) with constructs expressing Myc/DDK-tagged full-length human Granzyme A protein (hGranzyme A-Myc/DDK), Myc/DDK-tagged full-length human Granzyme B protein (hGranzyme B-Myc/DDK), Myc/DDK-tagged full-length human Granzyme H protein (hGranzyme H-Myc/DDK), Myc/DDK-tagged full-length human Granzyme K protein (hGranzyme K-Myc/DDK), and Myc/DDK-tagged full-length human Granzyme M protein (hGranzyme M-Myc/DDK), using Granzyme H (E4T2E) Rabbit mAb (upper), Myc-Tag (71D10) Rabbit mAb #2278 (middle), and β-Actin (D6A8) Rabbit mAb #8457 (lower).
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody product webpage for recommended antibody dilution.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised June 2020
Protocol Id: 10
Granzyme H (E4T2E) Rabbit mAb recognizes endogenous levels of total Granzyme H protein. This antibody does not cross-react with human Granzyme A, B, K, or M proteins.
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Gln195 of human Granzyme H protein.
Granzymes are a family of serine proteases expressed by cytotoxic T lymphocytes and natural killer (NK) cells and are key components of immune responses to pathogens and transformed cells (1). Granzymes are synthesized as zymogens and are processed into mature enzymes by cleavage of a leader sequence. They are released by exocytosis in lysosome-like granules containing perforin, a membrane pore-forming protein. Granzyme B has the strongest apoptotic activity of all the granzymes as a result of its caspase-like ability to cleave substrates at aspartic acid residues thereby activating procaspases directly and cleaving downstream caspase substrates (2,3).
Granzyme H has chymotrypsin-like thioester activity with a preference for hydrophobic, aromatic amino acid residues, such as phenylalanine, tyrosine, or methionine, at the P1 site (4,5). Granzyme H is predominantly expressed at high levels in NK cells, but not in T lymphocytes, and has also been described in mast cells (6-8). After perforin-mediated entry into a target cell, Granzyme H induces many hallmarks of programmed cell death, such as mitochondrial depolarization, generation of reactive oxygen species, DNA degradation, and chromatin condensation (9,10).
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