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| Product Name | Pyruvate Dehydrogenase (PDH) Activity Microplate Assay Kit |
| Catalog No. | CMTR-HMM-0081 |
| Description | Colorimetric microplate assay for quantitative measurement of pyruvate dehydrogenase (PDH) enzyme activity in isolated mitochondria, permeabilized cells, and tissue homogenates. PDH is the rate-limiting enzyme linking glycolysis to the TCA cycle by catalyzing the irreversible oxidative decarboxylation of pyruvate to acetyl-CoA with reduction of NAD+ to NADH. The assay couples PDH-produced NADH to the reduction of a tetrazolium dye (WST-1) via an electron mediator, producing a colored formazan product (absorbance 450 nm) proportional to PDH activity. An included PDH enzyme standard enables calculation of absolute activity units (mU/mg protein). |
| Intended Use | Quantitative measurement of PDH enzyme activity for studies of glucose metabolism, pyruvate dehydrogenase complex deficiency, cancer metabolism (Warburg effect), diabetes, and mitochondrial function assessment. |
| Principle / Technology | PDH: Pyruvate + CoA + NAD+ → Acetyl-CoA + CO2 + NADH; NADH + WST-1 (via electron mediator) → NAD+ + formazan (colored, A450); absorbance proportional to PDH activity; PDH standard enables absolute quantification. |
| Detection Method | Absorbance microplate reader at 450 nm (reference 620 nm); kinetic measurement at 1-minute intervals for 10-30 minutes at 30 °C. |
| Sample Type | Isolated mitochondria, digitonin-permeabilized cultured cells, fresh tissue homogenates (liver, heart, skeletal muscle, brain). |
| Performance Range / Specifications | PDH activity linear range: 0.1-10 mU/well; applicable to samples containing 1-50 µg mitochondrial protein; reaction linear for 20-30 minutes at 30 °C. |
| Sensitivity / LOD | Detection limit: 0.05 mU/well PDH activity; absorbance change >0.01 AU/min detectable. |
| Specificity | Specific for PDH complex activity; <5% contribution from α-ketoglutarate dehydrogenase or branched-chain α-keto acid dehydrogenase at recommended pyruvate concentration (5 mM). |
| Reaction Conditions / Protocol | Add sample/standard to plate; add reaction mix (pyruvate, CoA, NAD+, WST-1, electron mediator); incubate at 30 °C in plate reader; record A450 at 1-min intervals for 20 min; calculate rate from linear portion; compare to PDH standard curve. |
| Components / Formulation | PDH standard (purified porcine heart, 100 mU), pyruvate substrate, coenzyme A, NAD+, WST-1/electron mediator mix, assay buffer, mitochondrial extraction buffer, digitonin permeabilization solution, protocol. |
| Storage Conditions | Store PDH standard at -80 °C; substrates and dyes at -20 °C; buffers at 2-8 °C. |
| Shelf Life | 6 months from date of manufacture. |
| Package Specifications | 100 tests, 400 tests. |
| Product Form | Lyophilized PDH standard and substrates; liquid buffers. |
| Quality Control | Each lot tested for PDH standard activity verification; substrate specificity; linearity of absorbance change with PDH concentration (R² >0.99). |
| Key Features | Direct PDH activity measurement; PDH standard included for absolute quantification; WST-1 colorimetric detection at 450 nm; kinetic mode for rate calculation; mitochondrial extraction protocol included; clinically relevant enzyme target. |
| Purity | PDH standard specific activity 5-15 U/mg; CoA ≥85% purity; NAD+ ≥97%. |
| Concentration | PDH standard: 100 mU/vial; reconstitute and serially dilute for standard curve. |
| Activity / Unit Definition | PDH standard: 1 unit produces 1 µmol NADH per minute at pH 7.8, 30 °C. |
| Molecular Weight | PDH complex: ~9.5 MDa (multi-subunit complex); pyruvate: 88.06 g/mol. |
| Source / Origin | PDH standard purified from porcine heart mitochondria; synthetic substrates and dyes. |
| pH Range / Optimal pH | Assay buffer pH 7.8; optimal PDH activity at pH 7.8-8.0. |
| Shipping Conditions | Cold pack (PDH standard on dry ice required). |
| Expiration Date / Stability | 6 months at -80 °C (PDH standard); other components 6 months at -20 °C. |
| Regulatory / Compliance | For research use only; not for diagnostic procedures. |
| Compatibility | Compatible with mitochondrial preparations from mammalian tissues (liver, heart, muscle, brain). Sample preparation must include protease inhibitors to prevent PDH degradation. PDH is cold-labile — keep all samples and reagents at 4 °C or above during processing; never freeze mitochondria before assay. |
| Recommended Buffer System | Potassium phosphate buffer with MgCl2 and thiamine pyrophosphate (TPP), pH 7.8. |
| Application Notes / Precautions | PDH activity is regulated by phosphorylation (inactive) and dephosphorylation (active). Include dichloroacetate (DCA, 1 mM) to inhibit PDH kinase and maximize measured activity if assessing total PDH capacity. Also include PDH kinase inhibitor-free control for basal activity measurement. Normalize activity to citrate synthase activity or mitochondrial protein content. PDH is cold-labile — do not freeze mitochondria before assay, process fresh tissue within 2 hours. |
| Batch-to-Batch Consistency | PDH standard activity within ±20% of specification; WST-1 reduction rate per nmol NADH within ±15% of reference lot. |
For research use only, not for clinical use.
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