Western blot analysis of extracts from various cell lines using Nectin-4/PVRL4 Antibody (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower). As expected, Nectin-4/PVRL4 expression is not detected in either MDA-MB-231 or A-549 cells.
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing Myc/DDK-tagged full-length human Nectin-4/PVRL4 protein (hNectin-4/PVRL4-Myc/DDK; +), using Nectin-4/PVRL4 Antibody (upper) and DYKDDDDK Tag Antibody #2368 (lower).
Western blot analysis of extracts from HT-1376 cells, untreated (-) or treated with PNGase F (+), using Nectin-4/PVRL4 Antibody.
|MW (kDa)||62, 75|
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 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
Nectin-4/PVRL4 Antibody recognizes endogenous levels of total Nectin-4/PVRL4 protein. Based upon sequence alignment, this antibody is not predicted to cross-react with PVR, PVRL1, PVRL2, and PVRL3 proteins.Species Reactivity:
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues near the amino terminus of human Nectin-4/PVRL4 protein. Antibodies are purified by protein A and peptide affinity chromatography.
Nectin-4/Poliovirus Receptor-Like 4 (PVRL4) is a type-I transmembrane glycoprotein that belongs to the immunoglobulin superfamily and promotes cell-cell adhesion by serving as a major component of adherens junctions (1-3). The extracellular domain of Nectin-4, which contains an Ig variable-like domain (V) and two Ig constant-like domains (C), mediates binding to the measles virus (4) and to neighboring cells through trans heterophilic interactions with Nectin-1 (5,6). Unlike other nectin family members, which are widely expressed in adult tissues, Nectin-4 expression in humans is largely restricted to the placenta (7). Research studies have demostrated that Nectin-4 is overexpressed in a variety of human solid tumors of the pancreas (8), breast (9,10), lung (11), and ovary (12). Due to its restricted expression pattern in normal human tissues, Nectin-4 may serve as a novel diagnostic and therapeutic target for a variety of human tumors.
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