Recombinant Human Transglutaminase 3/TGM3 Protein, CF

• 6 months from date of receipt, -20 to -70 °C as supplied.
• 3 months, -20 to -70 °C under sterile conditions after opening.

• Assay Buffer: 50 mM Tris, 10 mM CaCl₂, 0.15 M NaCl, 0.05% Brij, pH 7.5 (TCNB)
• Assay Diluent: Deionized water
• Recombinant Human Transglutaminase 3/TGM3 (rhTGM3) (Catalog # 4604-TG)
• Bacterial Thermolysin (Thermolysin) (Catalog # 3097-ZN)
• Substrate: Z-Gln-Gly (Sigma, Catalog # C6154), dissolve 500 mM in deionized water, then adjust to pH 9.0 with NaOH
• MES, pH 6.0, 400 mM stock in deionized water
• DTT (Sigma, Catalog # D-0632), 200 mM stock in deionized water
• CaCl₂, 200 mM stock in deionized water
• 1 M Hydroxylamine Hydrochloride (Sigma, Catalog # 159417), dissolve in deionized water, then adjust 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. Combine the following volumes per reaction vial: 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, and 10 µL of 1.0 M Hydroxylamine Hydrochloride right before running assays. 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 rhTGM3 to 0.2 mg/mL with 2 ng/µL Thermolysin in Assay Buffer.
3. Incubate 30 minutes at room temperature.
4. Dilute rhTGM3 to 0.05 mg/mL in Assay Diluent.
5. Mix 20 µL of the diluted rhTGM3 with 80 µL Substrate Mixture. For the Blank mix 20 µL of Assay Diluent with 80 µL Substrate Mixture.
6. Incubate at 37 °C for 2 hours.
7. After incubation, stop the reaction with 400 µL of the Stop Solution. Mix well.
8. Centrifuge at top speed for 2 minutes in a microcentrifuge.
9. Load 200 µL of the supernatant into a plate.
10. Read at 525 nm (absorbance) in endpoint mode.
11. Calculate specific activity:

     *Adjusted for Substrate Blank

     **Derived using calibration standard L-glutamic acid g-monohydroxamate (Sigma, Catalog # G2253).

• rhTGM3: 0.4 µg
• Substrate: 10 mM