Western blot analysis of extracts from mouse brain, rat brain and SH-SY5Y cells using β2-Chimerin (2E3) Rat mAb.
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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.
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: 340
β2-Chimerin (2E3) Rat mAb recognizes endogenous levels of total β2-chimerin protein.Species Reactivity:
Human, Mouse, Rat
Monoclonal antibody is produced by immunizing animals with full-length recombinant human β2-Chimerin.
Chimerins are a family of GTPase-activating proteins (GAPS) that facilitate GTP hydrolysis by the small GTPase Rac, rendering it inactive and regulating cell shape, spreading and motility. Regulation of chimerin proteins occurs in response to growth factor receptor or G-protein coupled receptor activation followed by phospholipase C activation. Chimerins are among the growing number of phorbol ester and diacylglycerol (DAG) effector molecules that do not belong to the PKC family of isoenzymes (reviewed in 1,2). β2-chimerin is highly expressed in brain and pancreas, and its expression is down-regulated in malignant gliomas (3). β2-chimerin is also down-regulated in breast cancer, and its expression causes GAP activity-dependent cell cycle arrest in MCF-7 breast cancer cells (4). Signaling from the epidermal growth factor receptor (EGFR) activates β2-chimerin and allows its association with Rac1 at the plasma membrane (5). Also in response to EGF, diacylglycerol kinase (DGK) γ interacts with β2-chimerin, promotes its translocation to the plasma membrane, and regulate its GAP activity (6).
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