NAD-Isocitrate Dehydrogenase (NAD-IDH) Activity Assay Kit

$ 323⁹⁵

NAD-Isocitrate Dehydrogenase (NAD-IDH) Activity Assay Kit

SKU #	E-BC-K651-M
Detection Instrument	Microplate reader (440-460 nm, optimum wavelength: 450 nm)

Product Details

Properties

Synonyms	NAD-IDH
Sample type	Animal tissue
Sensitivity	0.84 U/L
Detection range	0.84-50 U/L
Assay type	Enzyme Activity
Assay time	50 min
Precision	Average inter-assay CV: 8.600% \| Average intra-assay CV: 5%
Other instruments required	Centrifuge, 37℃ incubator
Storage	-20℃
Valid period	12 months

Dilution of Sample

The recommended dilution factor for different samples is as follows (for reference only):

Sample type	Dilution factor
10% Rat kidney tissue homogenate	1
10% Mouse brain tissue homogenate	1
10% Mouse kidney tissue homogenate	1
10% Mouse heart tissue homogenate	1
10% Mouse liver tissue homogenate	1
10% Rat lung tissue homogenate	1

Note: The diluent is buffer solution. For the dilution of other sample types, please do pretest to confirm the dilution factor.

Detection Principle

Isocitrate dehydrogenase (IDH) is one of the inverters in the tricarboxylic acid cycle and plays an important role in energy metabolism, amino acid and vitamin synthesis. The cofactors of this enzyme include two kinds of NAD+ and NADP+, which are located in different parts of the cell respectively. In eukaryotic cells, NAD-dependent isocitrate dehydrogenase mainly exists in mitochondria. Under the activation of the activator, IDH converts isocitrate into α-ketoglutaric acid. Meanwhile, NAD+ is reduced to NADH, which under the action of hydrogen transmitter, transfer electrons to WST-8 to produce the yellow product. The activity of NAD-IDH can be calculated by measuring the change of absorbance value at 450 nm.

Kit Components & Storage

Item	Component	Size (96 T)	Storage
Reagent 1	Buffer Solution	60 mL × 2 vials	-20℃, 12 months
Reagent 2	Substrate	1.6 mL × 1 vial	-20℃, 12 months, shading light
Reagent 3	Accelerant	Powder × 2 vials	-20℃, 12 months, shading light
Reagent 4	Chromogenic Agent	3 mL × 1 vial	-20℃, 12 months, shading light
Reagent 5	Standard	Powder × 2 vials	-20℃, 12 months, shading light
	Microplate	96 wells	No requirement
	Plate Sealer	2 pieces

Note: The reagents must be stored strictly according to the preservation conditions in the above table. The reagents in different kits cannot be mixed with each other. For a small volume of reagents, please centrifuge before use, so as not to obtain sufficient amount of reagents.

Technical Data:

Parameter:

Intra-assay Precision

Three rat kidney tissue samples were assayed in replicates of 20 to determine precision within an assay (CV = Coefficient of Variation).

Parameters	Sample 1	Sample 2	Sample 3
Mean (U/L)	2.60	18.40	38.50
%CV	5.4	4.9	4.7

Inter-assay Precision

Three rat kidney tissue samples were assayed 20 times in duplicate by three operators to determine precision between assays.

Parameters	Sample 1	Sample 2	Sample 3
Mean (U/L)	2.60	18.40	38.50
%CV	8.2	9.4	8.2

Recovery

Take three samples of high concentration, middle concentration and low concentration to test the samples of each concentration for 6 times parallelly to get the average recovery rate of 98%.

	Standard 1	Standard 2	Standard 3
Expected Conc. (mmol/L)	0.25	0.52	0.88
Observed Conc. (mmol/L)	0.2	0.5	0.9
Recovery rate (%)	94	98	99

Sensitivity

The analytical sensitivity of the assay is 0.84 U/L. This was determined by adding two standard deviations to the mean O.D. obtained when the zero standard was assayed 20 times, and calculating the corresponding concentration.

Standard Curve

As the OD value of the standard curve may vary according to the conditions of the actual assay performance (e.g. operator, pipetting technique or temperature effects), so the standard curve and data are provided as below for reference only:

Concentration (μmol/L)	0	0.2	0.3	0.4	0.6	0.8	0.9	1
Average OD	0.077	0.185	0.255	0.316	0.428	0.544	0.616	0.679
Absoluted OD	0	0.108	0.178	0.239	0.351	0.467	0.539	0.602