Recombinant Human BCAT1 Protein, CF Summary
with an 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, DTT and Glycerol.|
|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: 50 mM Tris, 0.05% Tween-20, pH 8.0
- Recombinant Human BCAT1 (rhBCAT1) (Catalog # 9536-BA)
- Recombinant Human NQO-1 (rhNQO-1) (Catalog # 7567-DH)
- Glutamate dehydrogenase (GIDH) (Sigma, Catalog # G7882), 200 U/mL sock in 50 mM Tris, 0.05% Tween-20, pH 8.0
- Nicotinamide adenine dinucleotide ( beta -NAD) (Sigma, Catalog # N6522), 100 mM stock in deionized water
- Resazurin (Catalog # AR002)
- alpha -Ketoglutaric Acid (Sigma, Catalog # K2010), 1 M stock in deionized water
- L-Leucine (EMD Biosciences, Inc., Catalog # 4330), 100 mM stock in deionized water
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute rhBCAT1 to 0.04 µg/mL in Assay Buffer.
- Dilute alpha -Ketoglutaric Acid to 100 mM in Assay Buffer.
- Prepare Substrate Mixture containing 100 U/mL GIDH, 2 mM beta -NAD, 40 µM Resazurin, 1 mM alpha -Ketoglutaric Acid, 4 mM L-Leucine, and 4 µg/mL rhNQO-1 in Assay Buffer.
- Load 50 µL of 0.04 µg/mL rhBCAT-1 in a plate, and start the reaction by adding 50 µL of Substrate Mixture. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of Substrate Mixture.
- Read at excitation and emission wavelengths of 540 nm and 585 nm (top read), respectively, in kinetic mode for 8 minutes with a 3 minute lag time in kinetic mode.
- Calculate specific activity:
Specific Activity (pmol/min/µg) =
|Adjusted Vmax* (RFU/min) x Conversion Factor** (pmol/RFU)|
|amount of enzyme (µg)|
*Adjusted for Substrate Blank.
**Derived using calibration standard Resorufin (Sigma, Catalog # R3257).
- rhBCAT1: 0.002 µg
- GIDH: 5 U
- beta -NAD: 1 mM
- rhNQO-1: 0.2 µg
- Resazurin: 0.02 mM
- alpha -Ketoglutaric Acid: 0.5 mM
- L-Leucine: 2 mM
Recombinant Human BCAT1 (Catalog # 9536-BA) is measured by its ability to convert leucine and alpha-ketoglutarate to alpha-ketoisocaproate and glutamate.
Branched-chain-amino-acid aminotransferases (BCATs) are enzymes that catalyze the first reaction in the catabolism of the essential branched-chain amino acids leucine, isoleucine, and valine to their respective keto-acids while concurrently producing glutamate. BCATs belong to the class-IV pyridoxal-phosphate-dependent (PLP-dependent) aminotransferase family of enzymes (1). There are two BCAT isozymes in humans and mammals, a mitochondrial form known as BCATm or BCAT2 and a cytosolic form known as BCATc or BCAT1 that share 55% sequence identity. In humans and rodents, BCATm is found in most tissues whereas BCATc is almost exclusively present in the nervous system (2,3) where it is thought to play a role in glutamate neurotransmitter metabolism (1, 4). The 43 kDa human BCATc exists as an active homodimer and has a unique CXXC active site near the dimerization domain (4). BCATc can be regulated by redox and is implicated as a marker for oxidative stress (5,6) and linked to Alzheimers Disease and Dementia (7). BCATc has been shown to cause cell proliferation (8) and significant evidence has been found implicating BCATc in several types of cancer (8-10). In many reports, BCATc can be further used as a marker or for diagnostic purposes in cancers (11‑13).
- Hutson, S. (2001) Prog. Nucleic. Acid Res. Mol. Biol. 70:175
- Suryawan, A. et al. (1998) Am. J. Clin. Nutr. 68:72.
- Hall, T.R. et al. (1993) J. Biol. Chem. 268:3092.
- Yennawar, N. H. (2006) J. Biol. Chem. 281:39660.
- Coles, S.J. et al. (2012) Acta.Biochim. Biophys. Sin. 44:172.
- El Hindy, M. et al. (2014) Antioxid. Redox Signal 20:2497.
- Ashby, E.L. et al. (2017) Neurochem. Res. 42:306.
- Zhang, L. and J. Han (2017) Biochem. Biophys. Res. Commun. 486:224.
- Zhu, W. et al. (2017) Mol. Carcinog. 56:1570.
- Zheng, Y.H. et al. (2016) Liver Int. 36:1836.
- Young, G.P. et al. (2016) Cancer Med. 5:2763.
- Pedersen, S.K. (2015) BMC Cancer. 15:654.
- Diaz-Lagares, A. et al. (2016) Clin. Cancer Res. 22:3361.
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