Recombinant Human Isocitrate Dehydrogenase 1/IDH1, CF

Catalog # Availability Size / Price Qty
7049-DH-010
Product Details
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Recombinant Human Isocitrate Dehydrogenase 1/IDH1, CF Summary

Purity
>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
Endotoxin Level
<1.0 EU per 1 μg of the protein by the LAL method.
Activity
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 #
N-terminal Sequence
Analysis
Ser2
Predicted Molecular Mass
47 kDa
SDS-PAGE
43-45 kDa, reducing conditions.

Product Datasheets

Carrier Free

What does CF mean?

CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.

What formulation is right for me?

In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.

7049-DH

Formulation Supplied as a 0.2 μm filtered solution in Tris, NaCl, Glycerol, Brij-35 and DTT.
Shipping The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.
Stability & 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.

Assay Procedure

Materials
  • 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 1012 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
Reconstitution Calculator

Reconstitution Calculator

The reconstitution calculator allows you to quickly calculate the volume of a reagent to reconstitute your vial. Simply enter the mass of reagent and the target concentration and the calculator will determine the rest.

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Background: Isocitrate Dehydrogenase 1/IDH1

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).

 

References
  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.
Entrez Gene IDs
3417 (Human); 15926 (Mouse); 24479 (Rat)
Alternate Names
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

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