Western blot analysis of extracts from various cell lines using ENPP3/CD203c (E5M2W) Rabbit mAb.
Western blot analysis of extracts from KU812 cells, untreated (-) or treated with PNGaseF (+), using ENPP3/CD203c (E5M2W) 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
ENPP3/CD203c (E5M2W) Rabbit mAb recognizes endogenous levels of total ENPP3/CD203c protein.Species Reactivity:
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human ENPP3/CD203c protein.
Ectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3/CD203c/PD-Ιbeta) is a type-II transmembrane glycoprotein that contains a large extracellular domain, an α-helical transmembrane segment, and a short cytoplasmic domain containing the N-terminus. ENPP3 has been shown to be overexpressed in colon carcinoma and is thought to play a role in tumor initiation and tumor cell invasiveness (1-3). Within the hematopoietic cell compartment, ENPP3 is a cell surface marker of basophil and mast cell lineages (4,5). Indeed, ENPP3 is overexpressed on transformed mast cells in patients with systemic mastocytosis (6). Recently, ENPP3 has been identified as being highly overexpressed in renal cell carcinoma (RCC) and may have potential as a novel therapeutic target for this disease (7).
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