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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. |
Background
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
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