Recombinant Human IMP Dehydrogenase 1/IMPDH1 Protein, CF Summary
Glu2-Tyr563, with a N-terminal Met and 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, EDTA, Glycerol and TCEP.|
|Shipping||The product is shipped with polar packs. 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: 50 mM Tris, 300 mM NaCl, pH 8.0
- Recombinant Human IMP Dehydrogenase 1/IMPDH1 (rhIMPDH1) (Catalog # 8904-DH)
- Nicotinamide adenine dinucleotide sodium salt ( beta -NAD) (Sigma, Catalog # N6522), 100 mM stock in deionized water
- Inosine 5'-monophosphate (IMP) (Sigma, Catalog # I4625), 100 mM stock in deionized water
- UV plate (Costar, Catalog # 3635)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute rhIMPDH1 to 20 μg/mL in Assay Buffer.
- Create Substrate Mixture containing 200 µM IMP and 400 µM beta -NAD in Assay Buffer.
- Load 50 μL of 20 μg/mL rhIMPDH1 into a plate, and start the reaction by adding 50 μL of Substrate Mixture. For Substrate Blank, load 50 μL of Assay Buffer and 50 μL of Substrate Mixture.
- 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 6220 M-1cm-1
***Using the path correction 0.320 cm
Note: the output of many spectrophotometers is in mOD Per Well:
- rhIMPDH1: 1.0 μg
- beta -NAD: 200 µM
- IMP: 100 µM
Background: IMP Dehydrogenase 1/IMPDH1
IMPDH1 (inosine monophosphate dehydrogenase) is a ubiquitous cytosolic and nuclear enzyme that plays a central role in guanine nucleotide metabolism. It catalyzes the NAD-dependent conversion of inosine monophosphate (IMP) to hypoxanthine monophosphate (XMP) which is a precursor for the synthesis of GMP, guanosine, and guanine. These compounds are critical for DNA synthesis and cell proliferation (1, 2). IMPDH1 associates into a homotetramer of approximately 55 kDa subunits, and the tetramers can aggregate into perinuclear structures (3, 4). IMPDH1 binds to the nucleotides AMP, ATP, IMP, and XMP (4) as well as to single stranded DNA and RNA (5). It is inhibited by the immunosuppressant drug mycophenolic acid (MPA) (3, 6) which induces reversible IMPDH1 aggregation (7). It is required for vascular endothelial cell proliferation and the terminal differentiation of adipocytes (8, 9). Mutations of IMPDH1 are associated with autosomal dominant retinitis pigmentosa (4, 10). Human IMPDH1 shares 98% amino acid sequence identity with mouse and rat IMPDH1. Alternative splicing generates additional isoforms with internal deletions or variable substitutions at the N-terminus.
- Lane, A.N. and T.W. Fan (2015) Nucleic Acids Res. 43:2466.
- Jackson, R.C. et al. (1975) Nature 256:331.
- Carr, S.F. et al. (1993) J. Biol. Chem. 268:27286.
- Thomas, E.C. et al. (2012) PLoS One 12:e51096.
- McLean, J.E. et al. (2004) Biochem. J. 379:243.
- Sintchak, M.D. et al. (1996) Cell 85:921.
- Ji, Y. et al. (2006) J. Biol. Chem. 281:206.
- Chong, C.R. et al. (2006) J. Med. Chem. 49:2677.
- Su, H. et al. (2009) Biochem. Biophys. Res. Commun. 386:351.
- Bowne, S.J. et al. (2002) Hum. Mol. Genet. 11:559.
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