Western blot analysis of extracts from 293 cells, mock transfected (-) or transfected with a construct expressing Myc-tagged full-length human Myc-tagged P2X7 receptor protein (hP2X7-Myc; +), and UACC-62 cells, using P2X7 Receptor (E1E8T) Rabbit mAb.
Immunoprecipitation of P2X7 Receptor from UACC-62 cell extracts using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or P2X7 Receptor (E1E8T) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using P2X7 Receptor (E1E8T) 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 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
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Protocol Id: 28
P2X7 Receptor (E1E8T) Rabbit mAb recognizes endogenous levels of total P2X7 receptor protein.Species Reactivity:
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Leu462 of human P2X7 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).
Purinoceptor 7 (P2X7) is a homotrimer involved in diverse cellular responses, including inflammation mediated by phospholipase A2, phospholipase D, MAP kinase, and NF-κB activation (3,4). Research studies suggest that P2X7 receptors promote apoptosis by regulating release of IL-1β in neurodegenerative disorders associated with inflammation (5). Microglial P2X7 receptors may contribute to neuroinflammatory responses in the ATP-rich site of neuronal injury (6) and mediate inflammatory pain (7, 8). Association studies demonstrate a possible causal link between P2RX7 gene polymorphisms and susceptibility to bipolar affective disorder and major depressive disorder (9,10).
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