Recombinant Mouse Transglutaminase 2/TGM2 Protein, CF

• 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:

     *Adjusted for Substrate Blank
     **Derived using calibration standard L-glutamic acid gamma -monohydroxamate (Sigma, Catalog # G2253).

• rmTGM2: 0.8 µg
• Substrate: 10 mM