|H M||Endogenous||78||Rabbit IgG|
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
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.
RasGRP3 (C33A3) Rabbit mAb detects endogenous levels of total RasGRP3 protein.
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy teminus of human RasGRP3.
Lymphocyte activation occurs in part through activation of the Ras signaling pathway following lymphocyte receptor stimulation. The RasGRP family of guanine nucleotide exchange factors (GEFs) catalyzes the exchange of GDP for GTP on Ras family small GTPases, promoting their active GTP-bound form. Diacylglycerol (DAG) or phorbol ester binding to RasGRP family members causes their translocation to the cell membrane and stimulates their activity (1,2). While T-Cells express RasGRP1, B-cells express both RasGRP1 and RasGRP3. RasGRP3 is important in linking B-cell receptor (BCR) activation to Ras signaling (3). In response to BCR stimulation, RasGRP3 is phosphorylated at Thr133 by PKC. This phosphorylation event further activates RasGRP3 in response to DAG, which stimulates PKC activity (4,5).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.
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|3334S||100 µl (10 western blots)||$ 255.0|