SKU: 4758334841

IDH1 His Tag Protein, Human

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Description

IDH1 His Tag Protein, HumanProduct 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

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.
2. Dang, L., et al. (2009). Cancer-associated IDH1 mutations produce 2-hydroxyglutarate. Nature, 462(7274), 739-744.
3. Xu, W., et al. (2011). Oncometabolite 2-hydroxyglutarate is a competitive inhibitor of α-ketoglutarate-dependent dioxygenases. Cancer Cell, 19(1), 17-30.

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 Buffer25 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)

SlopeAdjusted for Substrate Blank

ext. coeffUsing the extinction coefficient 6270 M-1cm-1

path corrUsing the path correction 0.320 cm

 

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SKU: 4758334841

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