Western blot analysis of extracts from COS-7 cells, untransfected or transfected with human SGK3, and untreated or treated with calf intestinal phosphatase (CIP), using Phospho-SGK3 (Thr320) (D30E6) Rabbit mAb (upper) or SGK3 Antibody #4227 (lower).
Western blot analysis of extracts from COS-7 cells, untransfected (-) or transfected with human SGK3 (+), using Phospho-SGK3 (Thr320) (D30E6) Rabbit mAb (upper) or SGK3 Antibody #4227 (lower).
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.
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-SGK3 (Thr320) (D30E6) Rabbit mAb detects overexpressed levels of SGK3 protein only when phosphorylated at Thr320.Species Reactivity:
HumanSpecies predicted to react based on 100% sequence homology:
Mouse, Rat, Monkey
Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr320 of human SGK3 protein.
Serum and glucocorticoid-inducible kinase (SGK) is a serine/threonine kinase closely related to Akt (1). SGK is rapidly induced in response to a variety of stimuli, including serum, glucocorticoid, follicle stimulating hormone, osmotic shock, and mineralocorticoids. SGK activation can be accomplished via HGF PI3K-dependent pathways and by integrin-mediated PI3K-independent pathways (2,3). Induction and activation of SGK has been implicated in activating the modulation of anti-apoptotic and cell cycle regulation (4-6). SGK also plays an important role in activating certain potassium, sodium, and chloride channels, suggesting its involvement in the regulation of processes such as cell survival, neuronal excitability, and renal sodium excretion (2). SGK is negatively regulated by ubiquitination and proteasome degradation (7).
SGK3 has been shown to be a downstream signaling molecule in the PI3K pathway. Its activation and phosphorylation at Thr320 by PDK1 may be an Akt-independent manner of signaling in cancer (8).
Explore pathways + proteins related to this product.
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