Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Fluorescein Conjugate) #6201 (-), SignalSilence® GSK-3α siRNA II (+) or SignalSilence® GSK-3α siRNA I #6312 (+), using GSK-3α Antibody #9338 and β-Actin (13E5) Rabbit mAb #4970. GSK-3α Antibody confirms silencing of GSK-3α expression and β-Actin (13E5) Rabbit mAb is used to control for loading and specificity of GSK-3α siRNA.
Western blot analysis of recombinant GST-GSK-3beta protein (76kD), and extracts from HeLa and PC3 cells, using GSK-3alpha Antibody (left) and GSK-3beta Antibody (right).
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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
GSK-3alpha Antibody detects endogenous levels of total GSK-3alpha protein. It does not cross-react with recombinant GSK-3beta.Species Reactivity:
Human, Mouse, Rat, Monkey
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to the sequence of human GSK-3alpha. Antibodies are purified by protein A and peptide affinity chromatography.
Glycogen synthase kinase-3 (GSK-3) was initially identified as an enzyme that regulates glycogen synthesis in response to insulin (1). GSK-3 is a ubiquitously expressed serine/threonine protein kinase that phosphorylates and inactivates glycogen synthase. GSK-3 is a critical downstream element of the PI3K/Akt cell survival pathway whose activity can be inhibited by Akt-mediated phosphorylation at Ser21 of GSK-3α and Ser9 of GSK-3β (2,3). GSK-3 has been implicated in the regulation of cell fate in Dictyostelium and is a component of the Wnt signaling pathway required for Drosophila, Xenopus, and mammalian development (4). GSK-3 has been shown to regulate cyclin D1 proteolysis and subcellular localization (5).
GSK-3alpha regulates the production of amyloid-beta peptides, a major component of the plaques that accumulate with progression of Alzheimer's disease. Administration of therapeutic concentrations of lithium, a GSK-3 inhibitor, attenuates amyloid-beta production by specifically inhibiting the cleavage of amyloid precursor protein (APP) by gamma secretase, blocking accumulation of amyloid-beta peptides in the brains of mice that overproduce APP (6).
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