Western blot analysis of extracts from various cell lines using Tryptase (E5F3Q) 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 Tryptase (Tryptase-Myc/DDK; +) or Myc/DDK-tagged full-length mouse Tryptase (Tryptase mouse-Myc/DDK; +), using Tryptase (E5F3Q) 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 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
Tryptase (E5F3Q) Rabbit mAb recognizes endogenous levels of total Tryptase protein. This antibody does not cross-react with murine Tryptase proteins.Species Reactivity:
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ala120 of human Tryptase protein.
Tryptase is the most abundant neutral serine protease expressed in the secretory granules of all human mast cells (1). Tryptase is secreted upon the coupled activation-degranulation response of mast cells in peripheral tissues to physical factors such as trauma, toxins, venoms, endogenous mediators, and immune mechanisms (IgE-dependent and IgE-independent) (2). Tryptase has distinct enzymatic functions that depend on the monomeric or homotetrameric state of this protein, the pH of the environment, and the presence or absence of heparin (3-5). Tryptase has the ability to cleave extracellular substrates such as vasoactive intestinal peptide (6), calcitonin gene-related peptide (7), fibronectin (8), fibrinogen (3), and kininogens (9). Tryptase is also a potent growth factor for epithelial cells, airway smooth muscle cells, and fibroblasts (10-13).
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