Recombinant Human Transglutaminase 7/TGM7 Protein, CF

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

• Recombinant Human Transglutaminase 7/TGM7 (rhTGM7) (Catalog # 5426-TG)
• Substrate: Z-Gln-Gly (Sigma, Catalog # C6154), 500 mM (dissolve in deionized water, then adjust to pH 9.0 with NaOH)
• 0.1 M MES, pH 6.0
• Dithiothreitol (DTT), 1 M stock in DMSO
• 1.0 M CaCl₂
• 1.0 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. Prepare substrate mixture right before running assay. Mix the following components per reaction:
1. 15 µL 500 mM Z-Gln-Gly
2. 75 µL 400 mM MES, pH 6.0
3. 7.5 µL 200 mM DTT
4. 7.5 µL 200 mM CaCl₂
5. 15 µL 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 assay. (For example: If there are 4 reaction vials, including blanks, multiply all volumes by 5)
2. Dilute rhTGM7 to 0.1 mg/mL in deionized water.
3. Mix 30 µL of the diluted rhTGM7 with 120 µL substrate mixture (step #1). Include a Substrate Blank containing 30 µL deionized water and 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. Transfer 200 µL (in duplicate) 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 g-monohydroxamate (Sigma, Catalog # G2253).

• rhTGM7: 0.8 µg
• Substrate: 10 mM