|H M R||Endogenous||60||Rabbit|
Western blot analysis of extracts from serum-starved COLO 201 cells, v-Src antibody immunoprecipitates from COLO 201 cells and PDGF-treated NIH/3T3 cells, treated with lambda phosphatase as indicated, using Phospho-Src (Tyr416) Antibody (upper) or v-Src antibody (lower).Learn more about how we get our images.
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
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.
Phospho-Src Family (Tyr416) Antibody detects endogenous levels of Src only when phosphorylated at tyrosine 416. The antibody may cross-react with other Src family members (Lyn, Fyn, Lck, Yes and Hck) when phosphorylated at equivalent sites. It does not cross-react with Src phosphorylated at tyrosine 527. It may cross react with phosphorylated RTKs.
Human, Mouse, Rat
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Tyr419 of human Src. Antibodies are purified by protein A and peptide affinity chromatography.
The Src family of protein tyrosine kinases, which includes Src, Lyn, Fyn, Yes, Lck, Blk, and Hck, are important in the regulation of growth and differentiation of eukaryotic cells (1). Src activity is regulated by tyrosine phosphorylation at two sites, but with opposing effects. While phosphorylation at Tyr416 in the activation loop of the kinase domain upregulates enzyme activity, phosphorylation at Tyr527 in the carboxy-terminal tail by Csk renders the enzyme less active (2).
Lyn is a member of the Src family that is predominantly expressed in hematopoietic cells (3). Lyn participates in signaling from multiple cell surface receptors such as the B cell antigen receptor (BCR) and CD40 (4).
Lck is essential for T-lymphocyte activation and differentiation (5,6). Phosphorylation of the carboxy-terminal Tyr505 downregulates Lck activity, while phosphorylation at Tyr394 leads to an increase in Lck activity (7).
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