Recombinant Mouse Transglutaminase 2/TGM2 Protein, CF

• Reactivity: Mu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Mouse Transglutaminase 2/TGM2 Protein, CF Summary

• Details of Functionality: Measured by its ability to form CBZ-Gln-Gly-Hydroxamate from CBZ-Gln-Gly and Hydroxylamine. The specific activity is >1,300 pmol/min/μg, as measured under the described conditions.
• Source: Spodoptera frugiperda, Sf 21 (baculovirus)-derived mouse Transglutaminase 2/TGM2 protein
  Ala2-Ala686, with an N-terminal Met and 6-His tag
• Accession #: P21981
• N-terminal Sequence: Met
• Protein/Peptide Type: Recombinant Enzymes
• Gene: Tgm2
• Purity: >90%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• 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: 78 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: 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 and Glycerol.
• Purity: >90%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
  • Assay Diluent: deionized water
  • Recombinant Mouse Transglutaminase 2/TGM2 (rmTGM2) (Catalog # 5418-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, 1.0 M stock
  • DTT (Sigma, Catalog # D-0632), 1.0 M stock in deionized water
  • CaCl₂,1.0 M stock in deionized water
  • 1.0 M Hydroxylamine Hydrochloride (Sigma, Catalog # 159417), dissolve in diH₂O, 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
  1. Prepare Substrate Mixture by combining right before running assays (Note: Dilute all components to the correct concentration in Assay Diluent.):
  1. 15 µL of 500 mM Substrate
  2. 75 µL of 400 mM MES, pH 6.0
  3. 7.5 µL of 200 mM DTT
  4. 7.5 µL of 200 mM CaCl₂
  5. 15 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).
  2. Dilute rmTGM2 to 0.1 mg/mL in Assay Diluent.
  3. Mix 30 µL of the diluted rmTGM2 with 120 µL Substrate Mixture. For the Substrate Blank mix 30 µL of Assay Diluent with 120 µL Substrate Mixture.
  4. Incubate at 37 °C for 2 hours.
  5. After incubation, stop the reaction with 600 µL of the Stop Solution. Mix well.
  6. Centrifuge at top speed for 2 minutes in a microcentrifuge.
  7. Load 200 µL of the supernatant into a plate.
  8. Read at 525 nm (absorbance) in endpoint mode.
  9. 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:
  • rmTGM2: 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 Mouse Transglutaminase 2/TGM2 Protein, CF

• C polypeptide
• EC 2.3.2.13
• G-ALPHA-h
• protein-glutamine gamma-glutamyltransferase 2
• protein-glutamine-gamma-glutamyltransferase
• TG(C)
• TG2
• TGase C
• TGase H
• TGase-2
• TGase-H
• TGC
• TGCGNAH
• TGM2
• Tissue transglutaminase
• transglutaminase 2 (C polypeptide, protein-glutamine-gamma-glutamyltransferase)
• Transglutaminase 2
• Transglutaminase C
• Transglutaminase H
• transglutaminase-2
• tTG

Background

Transglutaminase 2 (TG2), encoded by the Tgm2 gene, is also known as tissue Transglutaminase (tTG), Transglutaminase 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).

1. Gentile, V. et al. (1991) J. Biol. Chem. 266:478.
2. Chen, J.S.K. and Mehta K. (1999) Internat. J. Biochem. Cell Biol. 31:817.
3. Griffin, M. et al. (2002) Biochem. J. 368:377.
4. Lai, T.S. et al. (1998) J. Biol. Chem. 273:1776.
5. Mishra, S. et al. (2007) J. Biol. Chem. 282:18108.
6. Kim, S-Y. et al. (2002) Neurochem. Int. 40:85.
7. Lesort, M. et al. (2000) Prog. Neurobiol. 61:439.

Bioinformatics

• Gene Symbol: Tgm2
• Uniprot:
  • P21981()