2μg (R: reducing condition, N: non-reducing condition).
IDH1 His Tag Protein, Human
IDH1 His Tag Protein, Human
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Product Details
Product Details
Product Specification
| Species | Human |
| Synonyms | IDH1, PICD, IDP |
| Accession | O75874-1 |
| Amino Acid Sequence | Met 1 - Leu 414 with His Tag at the C-Terminus |
| Expression System | E.coli |
| Molecular Weight | 40-55kDa (Reducing) |
| Purity | ＞95% by SDS-PAGE,> 90% by HPLC |
| Conjugation | Unconjugated |
| Tag | His Tag |
| Physical Appearance | Liquid |
| Storage Buffer | 50mM Tris, 150mM NaCl, pH7.5, 1mM DTT, 10%Glycerol |
| Stability & Storage |
Stable for 12 months upon stored at -80℃ from the date of receipt. And avoid repeated freeze-thaws cycles. |
| Reference | 1. Parsons, D.W., et al. (2008). An integrated genomic analysis of human glioblastoma multiforme. Science, 321(5897), 1807-1812. |
Background
IDH1 (Isocitrate Dehydrogenase 1) is a key metabolic enzyme located in the cytoplasm and peroxisomes. Its primary physiological function is to catalyze the oxidative decarboxylation of isocitrate to produce alpha-ketoglutarate (α-KG), while concurrently reducing NADP+ to NADPH. This reaction is crucial for cellular metabolism, as it links the tricarboxylic acid (TCA) cycle with lipid metabolism and provides NADPH, a major reducing agent essential for antioxidant defense and anabolic biosynthesis.A seminal discovery in cancer biology revealed that specific heterozygous somatic mutations in the IDH1 gene, most commonly at the R132 residue, are frequent drivers in several cancers. These include gliomas (especially secondary glioblastoma and oligodendroglioma), acute myeloid leukemia (AML), cholangiocarcinoma, and chondrosarcomas. The mutant enzyme acquires a neomorphic activity: instead of producing α-KG, it reduces α-KG to D-2-hydroxyglutarate (D-2HG). This oncometabolite, D-2HG, accumulates to high levels and functions as a competitive inhibitor of multiple α-KG-dependent dioxygenases. This inhibition leads to profound epigenetic dysregulation (via inhibition of histone and DNA demethylases) and blockade of cellular differentiation, thereby promoting tumorigenesis.
Protocol
Assay protocol
Principle: Measured by the ability to oxidatively decarboxylate isocitrate to 2-oxoglutarate.
Materials
1. Assay Buffer：25 mM Tris, 0.5 mM MnCl2, 5 mM DTT, pH 7.5
2. IDH1 His Tag Protein, Human
3. Substrate1: DL-Isocitricacid (MCE, Catalog # HY-W009362)
4. Substrate2: NADP+ (Aladdin, Catalog # N101669)
5. 96 Well Clear Plate (BIOFIL, Catalog#011096)
6. Plate Reader (PerkinElmer, ABS,340 nm, kinetic mode,60s/cycle,10 cycles)
Produce
1. Dilute IDH1 to 0.8 μg/mL, 0.4 μg/mL 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 dilute IDH1 protein 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 10 minutes.
5. Calculate specific activity.
Specific Activity (pmol/min/µg) = Slope (OD/min) x well volume (L) x 1012pmol/mol
ext. coeff (M-1cm-1) x path corr. (cm) x amount of enzyme (μg)
Slope：Adjusted for Substrate Blank
ext. coeff：Using the extinction coefficient 6270 M-1cm-1
path corr：Using the path correction 0.320 cm
Picture
Picture
SDS-PAGE
SEC-HPLC
The purity of IDH1 His Tag Protein, Human is more than 90% determined by SEC-HPLC.
