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 K (E9I3O) Rabbit mAb (upper), Myc-Tag (71D10) Rabbit mAb #2278 (middle), and α-Actinin (D6F6) XP® Rabbit mAb #6487 (lower).
Western blot analysis of extracts from various cell lines using Granzyme K (E9I3O) Rabbit mAb (upper) 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 datasheet or 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 November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised October 2016
Protocol Id: 10
Granzyme K (E9I3O) Rabbit mAb recognizes endogenous levels of total Granzyme K protein. This antibody does not cross-react with human Granzyme A, B, H, or M proteins.Species Reactivity:
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Asp196 of human Granzyme K 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 K has tryptase-like activity with a preference for cleaving its specific substrates after basic residues, arginine or lysine (4-7). Granzyme K is expressed by NK T cells, cytotoxic T cells, and NK cells (8,9). After perforin-mediated entry into a target cell, Granzyme K induces apoptosis via generation of reactive oxygen species, DNA damage, and BID-dependent mitochondrial damage (10-12).
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