Recombinant Human Transglutaminase 1/TGM1 Protein, CF

Catalog # Availability Size / Price Qty
7765-TG-050
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Recombinant Human Transglutaminase 1/TGM1 Protein, CF Summary

Purity
>70%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
Endotoxin Level
<1.0 EU per 1 μg of the protein by the LAL method.
Activity
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 #
N-terminal Sequence
Analysis
Inconclusive. Gly6, Ser7, Thr491, Ala571 identified. Poly His detected by Western blot.
Predicted Molecular Mass
89 kDa
SDS-PAGE
75-88 kDa, reducing conditions

Product Datasheets

7765-TG

Formulation Supplied as a 0.2 μm filtered solution in Tris, NaCl, EDTA, 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.
  • 6 months from date of receipt, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.

Assay Procedure

Materials
  • 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
Reconstitution Calculator

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Background: Transglutaminase 1/TGM1

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).

References
  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.
Entrez Gene IDs
7051 (Human); 21816 (Mouse); 60335 (Rat)
Alternate Names
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

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