Western blot analysis of extracts from HeLa cells, untreated (-) or treated (+) with calf intestinal alkaline phosphatase (CIP)/λ phosphatase, and BaF3 cells, using Phospho-Pyruvate Dehydrogenase α1 (Ser293) Antibody (upper) or β-Actin (D6A8) Rabbit mAb #8457 (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 nonfat dry milk, 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.
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 June 2016
Protocol Id: 263
Phospho-Pyruvate Dehydrogenase α1 (Ser293) Antibody recognizes endogenous levels of pyruvate dehydrogenase α1 protein only when phosphorylated at Ser293 residue. Based on amino acid sequence comparisons, this antibody is predicted to detect endogenous levels of pyruvate dehydrogenase α2 protein only when phosphorylated at Ser291 residue.Species Reactivity:
Human, Mouse, Rat, Monkey
Polyclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser293 of human pyruvate dehydrogenase α1 protein.
The pyruvate dehydrogenase complex catalyzes the conversion of pyruvate and CoA into acetyl-CoA and CO2 in the presence of NAD+. Acetyl-CoA then goes into the citric acid cycle where it reacts with oxaloacetate to form citrate. Acetyl-CoA is also used for fatty acid and cholesterol biosynthesis. The reaction of oxidative decarboxylation of pyruvate therefore serves as a critical link between glycolysis and the citric acid cycle and lipid metabolism. In mammalian cells, the pyruvate dehydrogenase complex is located in the mitochondrial matrix (1). This complex is comprised of three enzymes: pyruvate dehydrogenase (E1), dihydrolipoamide acetyltransferase (E2) and dihydrolipoamide dehydrogenase (E3). Pyruvate dehydrogenase (E1) consists of two subunits: α and β. This enzyme catalyzes the removal of CO2 from pyruvate. Mutations in the α subunits of pyruvate dehydrogenase (E1) lead to congenital defects that are usually associated with lactic acidosis, neurodegeneration and early death (2).
Pyruvate dehydrogenase kinase 1 phosphorylates pyruvate dehydrogenase (E1) α1 subunit at Ser293 to inactivate its activity (3, 4). This phosphorylation contributes to the tumor metabolic reprogramming toward glycolysis in hypoxia by inhibiting the citric acid cycle (4).
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