Recombinant Human Transglutaminase 1/TGM1 Protein, CF

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

Recombinant Human Transglutaminase 1/TGM1 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 >100 pmol/min/μg, as measured under the described conditions.
• Source: Spodoptera frugiperda, Sf 21 (baculovirus)-derived human Transglutaminase 1/TGM1 protein
  Met2-Ala817, with an N-terminal Met and 6-His tag
• Accession #: P22735
• N-terminal Sequence: Inconclusive. Gly6, Ser7, Thr491, Ala571 identified. Poly His detected by Western blot.
• Protein/Peptide Type: Recombinant Enzymes
• Gene: TGM1
• Purity: >70%, 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: 89 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: 75-88 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, EDTA, DTT and Glycerol.
• Purity: >70%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
  • Assay Buffer: deionized water
  • Recombinant Human Transglutaminase 1/TGM1 (rhTGM1) (Catalog # 7765-TG)
  • Substrate: Z-Gln-Gly (Sigma, Catalog # C6154), 500 mM, pH 9.0 in deionized water
  • Hydroxylamine Hydrochloride (Sigma, Catalog # 159417), 1 M, pH 6.0 in deionized water
  • 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 containing 62.5 mM Substrate, 250 mM MES pH 6.0, 12.5 mM DTT, 12.5 mM CaCl₂, and 125 mM Hydroxylamine Hydrochloride.
  2. Dilute rhTGM1 to 0.2 mg/mL in Assay Buffer.
  3. Mix 30 µL dilute rhTGM1 with 120 µL Substrate Mixture. Include an Enzyme Control containing 30 µL Assay Buffer with 120 µL Substrate Mixture.
  4. Incubate at 37 °C for 2 hours.
  5. Stop each reaction with 600 µL of Stop Solution. Mix well.
  6. Centrifuge at top speed for 2 minutes in a microcentrifuge.
  7. Load 200 µL of the supernatant into a 96 well clear plate.
  8. Read plate 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 Enzyme Control
  **Derived using calibration standard L-glutamic acid gamma -monohydroxamate (Sigma, Catalog # G2253).

  Per Well:
  • rhTGM1: 1.6 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 1/TGM1 Protein, CF

• Epidermal TGase
• ICR2
• KTG
• LI
• LI1
• LI1EC 2.3.2.13
• protein-glutamine gamma-glutamyltransferase K
• protein-glutamine-gamma-glutamyltransferase)
• TG(K)
• TGASE
• TGase-1
• TGK
• TGKTGase K
• TGM1
• transglutaminase 1 (K polypeptide epidermal type I
• Transglutaminase 1
• Transglutaminase K
• transglutaminase, keratinocyte
• transglutaminase-1

Background

Transglutaminase 1, also known as TGM1, Transglutaminase K, keratinocyte transglutaminase, or epidermal TGase, is a member of the transglutaminase family. Transglutaminases catalyze the calcium-dependent formation of isopeptide bonds between lysine and glutamine residues (1). TGM1 plays an important role in formation of the cornified envelope of differentiating keratinocytes in the epidermis through its cross-linking of epidermal proteins (2). The majority of cases of lamellar ichthyosis result from mutations present within the TGM1 gene and the subsequent dysfunction of TGM1 (1, 3).

1. Lorand, L. and R.M. Graham (2003) Nature Rev. Mol. Cell. Biol. 4:140.
2. Eckert, R.L. et al. (2005) J. Invest. Dermatol. 124:481.
3. Farasat, S. et al. (2009) J. Med. Genet. 46:103.

Bioinformatics

• Gene Symbol: TGM1
• Uniprot:
  • P22735(Human)