Western blot analysis of extracts from MKN-45 cells, untreated or SU11274-treated (1 μM for 2 hr), using Phospho-Semaphorin 4B (Ser825) Antibody (upper) and Semaphorin 4B Antibody #3355 (lower).
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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.
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
Phospho-Semaphorin 4B (Ser825) Antibody detects endogenous levels of semaphorin 4B protein only when phosphorylated at Ser825.Species Reactivity:
Human, Mouse, Rat
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser825 of human semaphorin 4B protein. Antibodies are purified by protein A and peptide affinity chromatography.
The semaphorin family of proteins is involved in axon guidance, cell migration, angiogenesis, and immune response. Plexins and neuropilins bind with high affinity to semaphorins to mediate their functions. Semaphorins are divided into seven classes of secreted or membrane-bound proteins. Members of Class 4 semaphorins include 4A through 4G; semaphorin 4B is a membrane-associated protein (1,2). Semaphorin 4B binds to the CLCP1 receptor and regulates cell motility (3). Furthermore, semaphorin 4B, like many other semaphorins, has a PDZ domain-binding motif at the carboxy terminus that interacts with PSD95 and localizes to the post-synaptic membrane (4).
The phosphorylation site at Ser825 was identified at Cell Signaling Technology using PTMScan® Technology, a proprietary proteomic method for post-translational modification site discovery (5).
Explore pathways + proteins related to this product.
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