Western blot analysis of extracts from 32D Clone 3 cells, untreated (-) or treated with Human Granulocyte Colony Stimulating Factor (hG-CSF) #8930 (100 ng/mL, 72 hr; +), mouse bone marrow, and NIH/3T3 cells using Neutrophil Elastase Antibody (Rodent Reactive) (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).
Western blot analysis of extracts from 293T cells, transfected with a construct expressing Myc/DDK-tagged full-length mouse neutrophil elastase protein (mNE-Myc/DDK; +) or mock transfected (-), using Neutrophil Elastase Antibody (Rodent Reactive) (upper), DYKDDDDK Tag Antibody #2368 (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 and 50% glycerol. 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
Neutrophil Elastase Antibody (Rodent Reactive) recognizes endogenous levels of total mouse and rat neutrophil elastase protein. This antibody does not cross-react with endogenously expressed human neutrophil elastase protein.
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Val100 of mouse neutrophil elastase protein. Antibodies are purified by protein A and peptide affinity chromatography.
Neutrophil elastase is hematopoietic serine protease that belongs to the chymotrypsin superfamily and plays a critical role in the innate immune function of mature neutrophils and monocytes (1,2). Neutrophil elastase is actively synthesized as an inactive zymogen in myelocytic precursor cells of the bone marrow, which then undergoes activation by limited proteolysis and sorting to primary (azurophil) storage granules of mature neutrophil granulocytes for regulated release (3,4). Research studies have shown that neutrophils play a significant role in mediating the inflammatory response through the release of neutrophil elastase, which activates pro-inflammatory cytokines and degrades components of the extracellular matrix and Gram-negative bacteria (5). Mutations in the gene encoding neutrophil elastase, ELA2, have been implicated in hematological diseases such as cyclic and severe congenital neutropenia, which is characterized by defects in promyelocyte maturation (6,7).
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