Western blot analysis of extracts from various cell lines using SAM68 Antibody.
|REACTIVITY||H M R Mk|
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
SAM68 Antibody recognizes endogenous levels of total SAM68 protein.Species Reactivity:
Human, Mouse, Rat, MonkeySpecies predicted to react based on 100% sequence homology:
Hamster, Bovine, Dog, Pig, Guinea Pig, Horse
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human SAM68 protein. Antibodies are purified by protein A and peptide affinity chromatography.
Src associated in mitosis 68 kDa (SAM68) is a member of the signal transduction and activation of RNA (STAR) family of RNA binding proteins (1,2). SAM68 is a multi-functional protein with roles in signal transduction, cell cycle regulation, transcription, and alternative splicing (1-3). SAM68 acts as a scaffold protein mediating responses to various stimuli that activate receptors, such as the T-cell receptor and the insulin receptor, linking activation of various signal transduction pathways with post-transcriptional gene regulation (3). SAM68 is a target for phosphorylation by Src-related kinases and Erk1/2 mitogen-activated protein kinases. Phosphorylation of SAM68 regulates both its affinity for RNA and alternative splicing of RNAs like CD44 receptor mRNA (1,4). Many of the activities of SAM68 and its targets for alternative splicing have been implicated in the development and progression of spinal muscular atrophy and cancers such as prostate and breast carcinomas, where SAM68 is frequently upregulated (1,5-8).
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