Recombinant Human Transglutaminase 4/TGM4 Protein, CF

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

Recombinant Human Transglutaminase 4/TGM4 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 >50 pmol/min/μg, as measured under the described conditions.
• Source: Spodoptera frugiperda, Sf 21 (baculovirus)-derived human Transglutaminase 4/TGM4 protein
  Met2-Lys684, with an N-terminal Met and 6-His tag
• Accession #: AAC50516
• N-terminal Sequence:

  No results obtained, Met predicted. Protein identity confirmed by MS analysis of tryptic fragments.

• Protein/Peptide Type: Recombinant Enzymes
• Gene: TGM4
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
• 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: 65-70 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, DTT and Glycerol.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
• Assay Procedure:
  • Assay Buffer: deionized water
  • Recombinant Human Transglutaminase 4/TGM4 (rhTGM4) (Catalog # 5760-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 sodium acetate pH 5.0, 12.5 mM DTT, 200 mM CaCl₂, 125 mM hydroxylamine hydrochloride, and 627 mM NaCl in deionized water.
  2. Dilute rhTGM4 to 0.2 mg/mL in Assay Buffer.
  3. Mix 30 µL dilute rhTGM4 with 120 µL Substrate Mixture. Include an enzyme blank 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 Substrate Blank
  **Derived using calibration standard L-glutamic acid gamma -monohydroxamate (Sigma, Catalog # G2253).

  Per Well:
  • rhTGM4: 1.6 µg
  • Substrate: 10 mM
  • Hydroxylamine: 20 mM

Notes

Coomassie is a registered trademark of Imperial Chemical Industries Ltd.

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human Transglutaminase 4/TGM4 Protein, CF

• EC 2.3.2.13
• Fibrinoligase
• hTGP
• Prostate transglutaminase
• Prostate-specific transglutaminase
• protein-glutamine gamma-glutamyltransferase 4
• TG(P)
• TGase P
• TGase-4
• TGM4
• TGP
• TGPFLJ26776
• transglutaminase 4 (prostate)
• Transglutaminase 4
• Transglutaminase P
• transglutaminase-4

Background

Transglutaminase 4, also known as TGM4, TG4, hTGP, or the prostate-specific transglutaminase, is a member of the transglutaminase family that is exclusively expressed and secreted from the prostate (1, 2). Transglutaminases catalyze the calcium-dependent formation of isopeptide bonds between lysine and glutamine residues (3). In rodents, TGM4 is responsible for the formation of the copulatory plug and sperm antigenicity, but the function in humans is largely unknown (4). The expression of TGM4 is primarily regulated by retinoic acid and secondarily modulated by androgens (5).

1. Dubbink, H.J. et al. (1996) Biochem. J. 315:901.
2. Dubbink, H.J. et al. (1999) Lab. Invest. 79:141.
3. Lorand, L. and Graham, R.M. (2003) Nature Rev. Mol. Cell. Biol. 4:140.
4. Williams-Ashman, H.G. et al. (1972) Proc. Natl. Acad. Sci. USA 69:2322.
5. Rivera-Gonzalez, G.C. et al. (2012) Nucleic Acids Res. 40:4825.
   
   

Bioinformatics

• Gene Symbol: TGM4
• Uniprot:
  • AAC50516()