Recombinant Human Isocitrate Dehydrogenase 1/IDH1, CF

• Reactivity: Hu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Human Isocitrate Dehydrogenase 1/IDH1, CF Summary

• Details of Functionality: Measured by the ability to oxidatively decarboxylate isocitrate to 2-oxoglutarate. The specific activity is >10,000 pmol/min/μg, as measured under the described conditions.
• Source: E. coli-derived human Isocitrate Dehydrogenase 1/IDH1 protein
  Met1-Leu414, with a C-terminal 6-His tag
• Accession #: O75874
• N-terminal Sequence: Ser2
• Protein/Peptide Type: Recombinant Enzymes
• Gene: IDH1
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 47 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 43-45 kDa, reducing conditions.

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris, NaCl, Glycerol, Brij-35 and DTT.
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
  • Assay Buffer: 25 mM Tris, 0.5 mM MnCl₂, 5 mM DTT, pH 7.5
  • Recombinant Human Isocitrate Dehydrogenase 1 (rhIDH1) (Catalog # 7049-DH)
  • DL-Isocitric acid (Sigma, Catalog # I1252), 100 mM stock in deionized water
  • NADP⁺ (Sigma, Catalog # N5755), 50 mM stock in deionized water
  • 96-well Clear Plate (Costar, Catalog # 2592)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rhIDH1 to 0.4 ng/μL in Assay Buffer.
  2. Prepare a Substrate Mixture by Diluting NADP⁺ and Isocitric Acid to 1 mM and 2 mM, respectively, in Assay Buffer.
  3. Load into a plate 50 μL of 0.4 ng/μL rhIDH1 and start the reaction by adding 50 μL of Substrate Mix. For Substrate Blanks, load 50 μL of Assay Buffer and 50 μL of Substrate Mix.
  4. Read plate at a wavelength of 340 nm (bottom read) in kinetic mode for 5 minutes.
  5. Calculate specific activity:

  Specific Activity (pmol/min/µg) = (Adjusted Vmax* (OD/min) x well volume (L) x 10^12 pmol/mol) / (ext. coeff** (M^-1cm^-1) x path corr.*** (cm) x amount of enzyme (µg))

  
  *Adjusted for Substrate Blank
  **Using the extinction coefficient 6270 M^-1cm^-1
  ***Using the path correction 0.32 cm
  Note: the output of many spectrophotometers is in mOD Per Well:
  • rhIDH1: 0.020 μg
  • NADP⁺: 0.5 mM
  • Isocitric Acid: 1 mM

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human Isocitrate Dehydrogenase 1/IDH1, CF

• Cytosolic NADP-isocitrate dehydrogenase
• EC 1.1.1.42
• IDCD
• IDH
• IDH1
• IDP
• IDPC
• isocitrate dehydrogenase [NADP] cytoplasmic
• isocitrate dehydrogenase 1 (NADP+), soluble
• Isocitrate Dehydrogenase 1
• NADP(+)-specific ICDH
• NADP-dependent isocitrate dehydrogenase, cytosolic
• NADP-dependent isocitrate dehydrogenase, peroxisomal
• Oxalosuccinate decarboxylase
• PICD

Background

Isocitrate Dehydrogenase 1 (IDH1) catalyzes the oxidative decarboxylation of isocitrate to alpha ‑ketoglutarate. There are two subclasses in the IDH family, one of them utilizing NADP⁺ as the electron acceptor and the other using NAD⁺ (1). The protein encoded by this gene is the NADP⁺-dependent isocitrate dehydrogenase found in the cytoplasm and peroxisomes. In peroxisomes, IDH1 generates the NADPH required for intraperoxisomal reduction reactions. Mutations of Arg132 of human IDH1 result in a reduced ability of the enzyme to convert isocitrate to alpha ‑ketoglutarate, but the enzyme acquires the ability to generate 2-hydroxyglutarate (2HG) from alpha ‑ketoglutarate (2). Elevated levels of the metabolite 2HG are associated with a high risk of malignant brain tumors. Arg132 mutations of IDH1 are common in high‑grade gliomas, but not in other types of tumors (3).

 

1. Nekrutenko, A. et al. (1998) Mol. Biol. Evol. 15:1674.
2. Dang, L. et al. (2009) Nature 462:739.
3. Bleeker, F.E. et al. (2009) Hum. Mutat. 30:7.

Bioinformatics

• Gene Symbol: IDH1
• Uniprot:
  • O75874()