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Isocitrate dehydrogenase activity colorimetric assay kit

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The Isocitrate Dehydrogenase Activity Colorimetric Assay Kit is a laboratory tool used to measure the activity of the enzyme isocitrate dehydrogenase. The kit provides a colorimetric-based method to quantify the enzymatic activity in samples.

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Lab products found in correlation

Synergy ht multi detection microplate reader, by Agilent Technologies (2 mentions) Multimode reader, by Agilent Technologies (1 mentions) Recombinant human idh1, by Abcam (1 mentions) Imark microplate reader, by Bio-Rad (1 mentions) Imarktm, by Bio-Rad (1 mentions)

7 protocols using isocitrate dehydrogenase activity colorimetric assay kit

1

Colorimetric Assay for IDH1 and IDH2

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The activities of IDH1 and IDH2 were detected using Isocitrate Dehydrogenase Activity Colorimetric Assay Kit according to the manufacturer’s instructions (BioVision, SanFrancisco, CA, USA). Briefly, recombinant human IDH1 (Abcam, Cambridge, UK) and IDH2 (Abcam, Cambridge, UK) were diluted with IDH assay buffer, and added into 96-well plate. And indicated concentration of TPL were added to these wells. Adjust the final volume to 50 µl with assay buffer. A total of 50 µl of the reaction mix, containing NADP+, IDH substrate, and developer, was added to each well. The mix was incubated for 3 min at 37 °C. The OD 450 nm was measured in a Bio-Tek multi-mode reader, and then the absorbance was read at 450 nm every 10 min, for 90 mins. The mean values of background wells without enzyme were subtracted from all readings.
Ye S., Xu P., Huang M., Chen X., Zeng S., Wang Q., Chen J., Li K., Gao W., Liu R., Liu J., Shao Y., Zhang H., Xu Y., Zhang Q., Zhong Z., Wei Z., Wang J., Hao B., Huang W, & Liu Q. (2020). The heterocyclic compound Tempol inhibits the growth of cancer cells by interfering with glutamine metabolism. Cell Death & Disease, 11(5), 312.
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2

Colorimetric Assay for IDH1 Activity

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To measure the production of NADH and NADPH, cIDH1-transfected cells (5×104) were processed using the Isocitrate Dehydrogenase Activity Colorimetric Assay Kit (BioVision) according to the manufacturer's instructions. The reaction mix was treated for 10 min, and the optical density at 450 nm was measured using an iMark microplate reader (Bio-Rad Laboratories).
Kawakami S., Michishita M., Sakaue M., Morimatsu M., Uemura M., Kashiwagi N., Maeda M., Machida Y., Azakami D., Egusa A.S., Onozawa E., Ishioka K., Watanabe M., Tanaka Y., Omi T, & Ochiai K. (2020). Novel canine isocitrate dehydrogenase 1 mutation Y208C attenuates dimerization ability. Oncology Letters, 20(6), 351.
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3

Measuring IDH Activity in Transfected Cells

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IDH activity was measured using an Isocitrate Dehydrogenase Activity Colorimetric Assay Kit (BioVision) as per the manufacturer’s protocol. Briefly, transfected HEK293FT cells were washed in ice cold PBS and homogenized in 200 μl IDH assay buffer. 50 μl of homogenized sample was transferred to one well of a 96-well plate, in triplicate, and mixed with 50 μl of assay reaction mix. The plate was incubated for 3 min at 37 °C and read at OD450 every 5 min for 2 h on a Synergy HT Multi-Detection Microplate Reader (Bio-Tek). Measurements were used to calculate IDH activity based on the generation of NADPH. For IDH competition assays, HEK293FT cells were co-transfected with BiFC-conjugated constructs along with increasing concentrations of HA-tagged wild type or mutant IDH1/2 and assayed for IDH activity as described above.
Robinson G.L., Philip B., Guthrie M.R., Cox J.E., Robinson J.P., VanBrocklin M.W, & Holmen S.L. (2016). In vitro visualization and characterization of wild type and mutant IDH homo- and heterodimers using Bimolecular Fluorescence Complementation. Cancer research frontiers, 2(2), 311-329.
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4

Colorimetric IDH Activity Assay

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IDH activity was determined using the commercial Isocitrate Dehydrogenase Activity Colorimetric Assay Kit (Biovision, Mountain View, CA, USA), according to the manufacturer's instructions. Briefly, NT2/D1 cells were lysed in an assay buffer provided in the kit. The lysate was centrifuged at 14,000 g for 15 min, and the cleared supernatant was used for the assay. NADP or NAD was used as the substrate for the NADP-IDH or NAD-IDH assay, respectively.
Yamada S., Kotake Y., Demizu Y., Kurihara M., Sekino Y, & Kanda Y. (2014). NAD-dependent isocitrate dehydrogenase as a novel target of tributyltin in human embryonic carcinoma cells. Scientific Reports, 4, 5952.
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5

Measuring IDH Activity in Transfected Cells

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IDH activity was measured using an Isocitrate Dehydrogenase Activity Colorimetric Assay Kit (BioVision) as per the manufacturer’s protocol. Briefly, transfected HEK293FT cells were washed in ice cold PBS and homogenized in 200 μl IDH assay buffer. 50 μl of homogenized sample was transferred to one well of a 96-well plate, in triplicate, and mixed with 50 μl of assay reaction mix. The plate was incubated for 3 min at 37 °C and read at OD450 every 5 min for 2 h on a Synergy HT Multi-Detection Microplate Reader (Bio-Tek). Measurements were used to calculate IDH activity based on the generation of NADPH. For IDH competition assays, HEK293FT cells were co-transfected with BiFC-conjugated constructs along with increasing concentrations of HA-tagged wild type or mutant IDH1/2 and assayed for IDH activity as described above.
Robinson G.L., Philip B., Guthrie M.R., Cox J.E., Robinson J.P., VanBrocklin M.W, & Holmen S.L. (2016). In vitro visualization and characterization of wild type and mutant IDH homo- and heterodimers using Bimolecular Fluorescence Complementation. Cancer research frontiers, 2(2), 311-329.
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6

Colorimetric Assay for NADH/NADPH

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To measure the production of NADH and NADPH, cIDH2-transfected cells (5 × 104 cells) were processed using the Isocitrate Dehydrogenase Activity Colorimetric Assay Kit (BioVision, Milpitas, CA, U.S.A.) as per
the manufacturer’s instructions. The reaction mix was treated for 10 min and then the optical density at 450 nm was measured using an iMarkTM microplate reader (BioRad, Hercules, CA, U.S.A.).
KAWAKAMI S., OCHIAI K., KATO Y., MICHISHITA M., HIRAMA H., OBARA R., AZAKAMI D., WATANABE M, & OMI T. (2017). Functional alteration of canine isocitrate dehydrogenase 2 (IDH2) via an R174K mutation. The Journal of Veterinary Medical Science, 80(1), 85-91.
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7

Colorimetric Assay of NAD-IDH Activity

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NAD-IDH activity was determined using the Isocitrate Dehydrogenase Activity Colorimetric Assay Kit (Biovision, Mountain View, CA, USA), according to the manufacturer's instructions. Briefly, NT2/D1 cells were lysed in an assay buffer provided in the kit. The lysate was centrifuged at 14,000 g for 15 min, and the cleared supernatant was used for the assay.
Asanagi M., Yamada S., Hirata N., Itagaki H., Kotake Y., Sekino Y, & Kanda Y. (2016). Tributyltin induces G2/M cell cycle arrest via NAD(+)-dependent isocitrate dehydrogenase in human embryonic carcinoma cells. The Journal of toxicological sciences, 41(2).
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