Recombinant Human Transglutaminase 2/TGM2 Protein, CF Summary
Details of Functionality
Measured by its ability to cleave a synthetic peptide Benzyloxycarbonyl-Gln-Gly and NH2OH. The specific activity is >1000 pmol/min/ug, as measured under the described condition.
Source
Spodoptera frugiperda, Sf 21 (baculovirus)-derived human Transglutaminase 2/TGM2 protein Ala2-Ala687 (Asn533Thr and Leu655Val), with an N-terminal Met and 6-His tag
>95%, by SDS-PAGE under reducing conditions and visualized by silver stain
Endotoxin Note
<1.0 EU per 1 μg of the protein by the LAL method.
Applications/Dilutions
Dilutions
Enzyme Activity
Theoretical MW
78 kDa. Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
SDS-PAGE
82 kDa, reducing conditions
Publications
Read Publications using 4376-TG in the following applications:
Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
6 months from date of receipt, -70 °C as supplied.
3 months, -70 °C under sterile conditions after opening.
Buffer
Supplied as a 0.2 μm filtered solution in Tris, NaCl, Glycerol and DTT.
Purity
>95%, by SDS-PAGE under reducing conditions and visualized by silver stain
Assay Procedure
Assay Diluent: deionized water
Recombinant Human Transglutaminase 2/TGM2 (rhTGM2) (Catalog # 4376-TG)
Substrate: Z-Gln-Gly (Sigma, Catalog # C6154), dissolve 500 mM in deionized water, then neutralize it to pH 9.0 with NaOH
MES, pH 6.0, 1 M stock
DTT (Sigma, Catalog # D-0632), 1 M stock in deionized water
CaCl2 ,1 M stock in deionized water
1 M Hydroxylamine Hydrochloride (Sigma, Catalog # 159417), dissolve in deionized H2O, then neutralize it to pH 6.0 with NaOH
Stop Solution: 0.37 M FeCl3 (Sigma, Catalog # 236489), 0.67 M HCl, 0.2 M Trichloroacetic Acid
96-well Clear Plate (Costar, Catalog # 92592)
Plate Reader (Model: Spectramax Plus by Molecular Devices) or equivalent
Prepare Substrate Mixture fresh before running assay. Mix the following components (Dilute all components to the correct concentration in Assay Diluent.):
10 µL of 500 mM Substrate
50 µL of 400 mM MES, pH 6.0
5 µL of 200 mM DTT
5 µL of 200 mM CaCl2
10 µL of 1 M Hydroxylamine Hydrochloride (Note: Multiply the volume for each component by the number of reaction vials + 1 to make enough substrate mixture for the assays. For example: If there are 2 reaction vials (including blank), multiply all volumes by 3.)
Dilute rhTGM2 to 0.1 mg/mL in Assay Diluent.
Mix 20 µL of the diluted rhTGM2 with 80 µL Substrate Mixture. For the Substrate Blank mix 20 µL of Assay Diluent with 80 µL Substrate Mixture.
Incubate at 37 °C for 2 hours.
After incubation, stop the reaction with 400 µL of the Stop Solution. Mix well.
Centrifuge at top speed for 2 minutes in a microcentrifuge.
Load 200 µL of the supernatant into a plate.
Read at 525 nm (absorbance) in endpoint mode.
Calculate specific activity:
Specific Activity (pmol/min/µg) =
Adjusted Abs* (OD) x Conversion Factor** (pmol/OD)
Incubation time (min) x amount of enzyme (µg)
*Adjusted for Substrate Blank **Derived using calibration standard L-glutamic acid gamma -monohydroxamate (Sigma, Catalog # G2253).
Per Well:
rhTGM2: 0.8 µg
Substrate: 10 mM
Notes
This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.
Alternate Names for Recombinant Human Transglutaminase 2/TGM2 Protein, CF
Transglutaminase 2 (TG2), encoded by the TGM2 gene, is also known as tissue transglutaminase (tTG), transgluytaminase C (TGC), and protein‑glutamine‑ gamma -glutamyltransferase. It belongs to the family of transglutaminases that catalyze the posttranslational modification of proteins via calcium dependent cross-linking reactions (1‑3). In addition to its function in protein cross-linking, TGM2 is also capable of hydrolyzing both GTP and ATP (4) and has intrinsic kinase activity (5). TGM2 has been implicated in a variety of human diseases including celiac disease, inclusion body myositis, atherosclerosis, and neurodegenerative diseases (6, 7).
Gentile, V. et al. (1991) J. Biol. Chem. 266:478.
Chen, J.S.K. and K. Mehta (1999) Internat. J. Biochem. Cell Biol. 31:817.
Griffin, M. et al. (2002) Biochem. J. 368:377.
Lai, T.S. et al. (1998) J. Biol. Chem. 273:1776.
Mishra, S. et al. (2007) J. Biol. Chem. 282:18108.
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