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Active Pyruvate Carboxylase (PC)

PCB

ACTIVITY TEST

Pyruvate carboxylase (PC) is a mitochondrial, biotin-containing enzyme catalyzing the ATP-dependent synthesis of oxaloacetate from pyruvate and bicarbonate, with a critical anaplerotic role in sustaining the brain metabolism. Pyruvate carboxylase (PC) deficiency is a rare autosomal recessive mitochondrial neurometabolic disorder of energy deficit resulting in high morbidity and mortality, with limited therapeutic options. Besides, PCK2 has been identified as an interactor of PC, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human PC and recombinant human PCK2. Briefly, biotin-linked PC were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100 ul were then transferred to PCK2-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST 3 times and incubation with Streptavidin-HRP for 30 min, then wells were aspirated and washed 5 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37℃. Finally, add 50 µl stop solution to the wells and read at 450 nm immediately. The binding activity of PC and PCK2 was shown in Figure 1, the EC50 for this effect is 0.056 ug/mL.

USAGE

Reconstitute in 10mM PBS (pH7.4) to a concentration of 0.1-1.0 mg/mL. Do not vortex.

STORAGE

Avoid repeated freeze/thaw cycles. Store at 2-8°C for one month. Aliquot and store at -80°C for 12 months.

STABILITY

The thermal stability is described by the loss rate. The loss rate was determined by accelerated thermal degradation test, that is, incubate the protein at 37°C for 48h, and no obvious degradation and precipitation were observed. The loss rate is less than 5% within the expiration date under appropriate storage condition.

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Magazine Citations
Chemico-Biological Interactions Treadmill exercise rescues mitochondrial function and motor behavior in the CAG140 knock-in mouse model of Huntington's disease Pubmed: 31778667
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