Microcalorimetry of Pyruvate Dehydrogenase (PDH) Activity Detection Kit

Pyruvate dehydrogenase（PDH）Instructions for Activity Detection Kit Micro method

Attention: The reagents of this product have been changed. Please pay attention to and strictly follow the instructions in this manual.

Item No.BC0385

Specifications100T/96S

Product composition: Before use, please carefully check whether the volume of the reagent matches the volume inside the bottle. If you have any questions, please contact the staff of Solaibao in a timely manner.

Microcalorimetry of Pyruvate Dehydrogenase (PDH) Activity Detection Kit

Microcalorimetry of Pyruvate Dehydrogenase (PDH) Activity Detection Kit

Preparation of solution:

1. Reagent 2：For volatile reagents, seal as soon as possible after use,-20℃preservation.

2. Preparation of working fluid: before use5.75mLReagent 4, one reagent 50.45mLReagent 61.75mLTransfer reagent seven to a bottle of reagent three and mix and dissolve for later use(In total)15.45mL, about85T）Unused reagents-20℃Packaging and preservation, can be saved4weekAvoid repeated freezing and thawing.

Product Description:

PDH（EC 4.1.1.1）Widely present in animals, plants, microorganisms, and cultured cells, it is a complex of pyruvate dehydrogenase(PDHC)The rate limiting enzyme that catalyzes the oxidation decarboxylation of pyruvic acid, catalyzing the de shuttle of pyruvic acidConnect glycolysis with the tricarboxylic acid cycle.

PDHCatalytic dehydrogenation of pyruvate and simultaneous reduction2,6-Dichlorophenol indophenol（2,6-DCPIP）As a result, it leads to605nmReduction in light absorption.

Pyruvic Acid + Thiamine Pyrophosphate Hydroxyethyl Thiamine Pyrophosphate

Dichlorophenolindophenol(605nm) + Hydroxyethyl Thiamine Pyrophosphate Reduced Dichlorophenolindophenol

Attention: It is recommended to choose before the experiment2-3Conduct preliminary experiments on samples with significant expected differences. If the absorbance value of the sample is not within the measurement range, it is recommended to dilute or increase the sample size for testing.

Equipment and supplies to be provided:

Visible spectrophotometer/ELISA reader, water bath, desktop centrifuge, adjustable pipette, micro glass colorimetric dish/96Orifice plate, mortar/Homogenizer, ice, and distilled water.

Operation steps:

1sample processing(The sample size to be tested can be adjusted appropriately, and the specific proportion can be referred to in the literature)

1. Organization: Weighing and Appointment0.1gOrganize, join1mLReagent 1 and10μLReagent 2: Grind the homogenate thoroughly using an ice bath homogenizer or mortar,4 ℃ 11000gcentrifugation10minTake the supernatant,Place it on ice and waitMeasure.

2. Cell or bacterial samples: Collect first500Transfer 10000 cells or bacteria into a centrifuge tube and discard the supernatant after centrifugation; Join later1mLReagent 1 and10μLReagent 2Ice bath ultrasonic crushing bacteria (power)200 W, Ultrasound3 s, interval7 s, total time5 min）； then11000g，4℃, centrifugation10 min，

Take the supernatant and place it on ice for testing.

3. Serum (plasma)And other liquid samples: Direct detection.If the solution is turbid, centrifuge and remove the supernatant for measurement.

2、 Measurement steps

1visiblePreheating of spectrophotometer or enzyme-linked immunosorbent assay reader30minAbove, adjust the wavelength to605nmAdjust the spectrophotometer to zero for distilled water.

2Each sample requires180μLWorking fluid,According to the experimental requirementsTake out a certain amount of working fluid and place it in37℃(Mammals) or25℃(Other species)In a water bath potwater bath10min.

3Blank tube: in a microcalorimeter or96Add to the orifice plate180μLWorking fluid and10μLWater, mix well, record immediately605nmplace10splacelight absorption valueA1and1min10sPost absorption valueA2, CalculateΔAblank=A1-A2.Blank tubes only need to be tested1-2time.

4Measurement tube: in a microcalorimeter or96Add to the orifice plate180μLWorking fluid and10μLSample, mix well, record immediately605nmplace10splacelight absorption valueA3and1min10sPost absorption valueA4, CalculateΔAdetermination=A3-A4.

IIIPDHActivity Calculation

a.The calculation formula for measuring with a microcalorimeter is as follows

1. Calculated based on sample protein concentration

Definition of Unit: EachmgHistone consumption per minute in the reaction system1nmol 2,6-Dichlorophenol indophenol is defined as an enzyme activity unit.

PDHActivity（U/mg prot）=[(ΔAdetermination-ΔAblank)÷（ε×d）×VAnti General×10⁹]÷(Cpr×Vsample) ÷T

=904.762×(ΔAdetermination-ΔAblank)÷Cpr

2. Calculated based on sample quality

Definition of Unit: EachgThe organization consumes every minute in the reaction system1nmol 2,6-Dichlorophenol indophenol is defined as an enzyme activity unit.

PDHActivity（U/gQuality)=[(ΔAdetermination-ΔAblank)÷（ε×d）×VAnti General×10⁹]÷(Vsample÷VSample General×W) ÷T

=913.81×(ΔAdetermination-ΔAblank)÷W

3. Calculated by the number of bacteria or cells

Definition of Unit: Each1Ten thousand bacteria or cells are consumed per minute in the reaction system1nmol 2,6-Dichlorophenol indophenol is defined as an enzyme activity unit.

PDHActivity（U/10⁴cell）=[(ΔAdetermination-ΔAblank)×VAnti General÷（ε×d）×10⁹]÷(Vsample÷VSample General×500)÷T

=1.828×(ΔAdetermination-ΔAblank)

4. Calculated by sample volume

Definition of Unit: EachmLliquidIn the reaction systemConsumption per minute1nmol 2,6-Dichlorophenol indophenol is defined as an enzyme activity unit.

PDHactivity（U/mL）=[(ΔAdetermination-ΔAblank)×VAnti General÷(ε×d)×10⁹]÷Vsample÷T

=904.762×(ΔAdetermination-ΔAblank)

VCounter total: the total volume of the reaction system,1.9×10^-4L；ε：2,6-Molar extinction coefficient of dichloroindolol,2.1×10⁴L/mol/cm；dColor dish optical diameter,1cm；VSample: Add sample volume,0.01mL；VSample total: Add the volume of reagent one and reagent two,1.01mL；TResponse time,1min；Cpr: Sample protein concentration,mg/mL；W: Sample quality,g；500Total number of bacteria or cells,500Wan.

b.use96The calculation formula for orifice plate measurement is as follows

1．Calculated based on sample protein concentration

Definition of Unit: EachmgHistone consumption per minute in the reaction system1nmol 2,6-Dichlorophenol indophenol is defined as an enzyme activity unit.

PDHActivity（U/mg prot）=[(ΔAdetermination-ΔAblank)×VAnti General÷（ε×d）×10⁹]÷(Cpr×Vsample) ÷T

=1809.524×(ΔAdetermination-ΔAblank)÷Cpr

2．Calculated based on sample quality

Definition of Unit: EachgThe organization consumes every minute in the reaction system1nmol 2,6-Dichlorophenol indophenol is defined as an enzyme activity unit.

PDHActivity（U/gQuality)=[(ΔAdetermination-ΔAblank)×VAnti General÷（ε×d）×10⁹]÷(Vsample÷VSample General×W) ÷T

=1827.62×(ΔAdetermination-ΔAblank)÷W

3．Calculated by the number of bacteria or cells

Definition of Unit: Each1Ten thousand bacteria or cells are consumed per minute in the reaction system1nmol 2,6-Dichlorophenol indophenol is defined as an enzyme activity unit.

PDHActivity（U/10⁴cell）=[(ΔAdetermination-ΔAblank)×VAnti General÷（ε×d）×10⁹]÷(Vsample÷VSample General×500) ÷T

=3.655×(ΔAdetermination-ΔAblank)

4．Calculated by sample volume

Definition of Unit: EachmLliquidIn the reaction systemConsumption per minute1nmol 2,6-Dichlorophenol indophenol is defined as an enzyme activity unit.

PDHactivity（U/mL）=[(ΔAdetermination-ΔAblank)×VAnti General÷(ε×d)×10⁹]÷Vsample÷T

=1809.524×(ΔAdetermination-ΔAblank)

VCounter total: the total volume of the reaction system,1.9×10^-4L；ε：2,6-Molar extinction coefficient of dichloroindolol,2.1×10⁴L/mol/ cm；d：96Aperture plate optical diameter,0.5cm；VSample: Add sample volume,0.01mL；VSample total: Add the volume of reagent one and reagent two,1.01mL；TResponse time,1min；Cpr: Sample protein concentration,mg/mL；W: Sample quality,g；500Total number of bacteria or cells,500Wan.

Notes:

1During the measurement process, all samples were placed on ice and2Detect within hours to avoid denaturation and inactivation.

2Measuring tubeΔAValue in0.01-0.25Between, if measuring the tubeΔAValue greater than0.25The sample needs to be diluted.Multiply the formula by the dilution factor.If measuring the tubeΔAvaluesmallYu0.01The sample size needs to be increased before retesting, and attention should be paid to synchronously modifying the calculation formula.

3Due to the presence of a certain concentration of protein in reagent one (approximately1mg/mL）Therefore, when measuring the protein concentration of the sample, it is necessary to subtractReagent 1The protein content.

Experimental Example:

1取0.1gMouse liverjoin1mLReagent 1 and10μLReagent 2: Grind the homogenate thoroughly using an ice bath homogenizer or mortar,4℃The11000gcentrifugation10minTake the supernatant and place it on ice. Follow the measurement steps and use itTrace quartz colorimetric dishmeasuredcalculateΔAdetermination=A3-A4=1.3106-1.1183=0.1923，ΔAblank=A1-A2 =1.3325-1.3314=0.0011Calculate enzyme activity based on sample quality to obtain:

PDHactivity(U/gquality)= 913.81×(ΔAdetermination-ΔAblank)÷W=1747 U/gquality

Related publications:

[1]Peng S, Wang Y, Zhou Y, et al. Rare ginsenosides ameliorate lipid overload-induced myocardial insulin resistance via modulating metabolic flexibility[J]. Phytomedicine, 2019, 58: 152745.

References:

[1]Guitart M, Andreu A L, García-Arumi E, et al. FATP1 localizes to mitochondria and enhances pyruvate dehydrogenase activity in skeletal myotubes[J]. Mitochondrion, 2009, 9(4): 266-272.

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