Western blot analysis of extracts from various cell lines using P2X4 Receptor (D9R1H) Rabbit mAb.
Western blot analysis of extracts from 293 cells, mock transfected (-) or transfected with a construct expressing Myc-tagged full-length human P2X4 receptor protein (hP2X4-Myc; +), using P2X4 Receptor (D9R1H) Rabbit mAb.
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 nonfat dry milk, 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.
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 June 2016
Protocol Id: 263
P2X4 Receptor (D9R1H) Rabbit mAb recognizes endogenous levels of total P2X4 receptor protein.Species Reactivity:
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro120 of human P2X4 receptor protein.
P2X purinergic receptors are ATP-gated ion channels involved in physiological processes that include inflammation, afferent sensory signaling, and sympathetic motor nerve activity. Seven different vertebrate genes (P2RX1-P2RX7) encode for individual receptor protein subunits (1). All P2X subunit proteins share similar protein domain structure, but can differ in overall protein length from 384 to 595 amino acids. Each P2X subunit is composed of amino- and carboxy-terminal intracellular domains, two transmembrane domains, and a large extracellular loop that contains ten evenly spaced cysteines and multiple glycosylation sites (2). P2X receptors are found in a variety of cell types and tissues, including central and peripheral nervous system neurons and glial cells, autonomic and sensory neurons, bone, muscle, and hematopoietic tissues (1).
P2X purinoceptor 4 (P2X4) trimers are expressed at the cell surface where they act as ligand-gated ion channels for monovalent and divalent cations (3). P2X4 receptors contribute to regulation of synaptic strength through participation in the formation of long-term potentiation (4,5). Research studies indicate that P2X4 receptor expression may play a role in influencing alcohol-drinking behavior and conferring protection to cardiac myocytes during heart failure (6,7). Additional studies show that microglial P2X4 receptors are upregulated during nerve-injury associated neuropathic pain (8).
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