Recombinant Human Isocitrate Dehydrogenase 1/IDH1, CF
Recombinant Human Isocitrate Dehydrogenase 1/IDH1, CF Summary
Met1-Leu414, with a C-terminal 6-His tag
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.
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.
|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.
- 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
- Dilute rhIDH1 to 0.4 ng/μL in Assay Buffer.
- Prepare a Substrate Mixture by Diluting NADP+ and Isocitric Acid to 1 mM and 2 mM, respectively, in Assay Buffer.
- 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.
- Read plate at a wavelength of 340 nm (bottom read) in kinetic mode for 5 minutes.
- 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
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).
- Nekrutenko, A. et al. (1998) Mol. Biol. Evol. 15:1674.
- Dang, L. et al. (2009) Nature 462:739.
- Bleeker, F.E. et al. (2009) Hum. Mutat. 30:7.
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