Recombinant Human Transglutaminase 3/TGM3 Protein, CF Summary
Ala2-Glu693 (Gly654Arg), with an N-terminal Met and 6-His tag
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
|Formulation||Supplied as a 0.2 μm filtered solution in Tris, NaCl and Glycerol.|
|Shipping||The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.|
|Stability & Storage:||Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
- 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
- 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.
- Dilute rhTGM3 to 0.2 mg/mL with 2 ng/µL Thermolysin in Assay Buffer.
- Incubate 30 minutes at room temperature.
- Dilute rhTGM3 to 0.05 mg/mL in Assay Diluent.
- 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.
- Incubate at 37 °C for 2 hours.
- After incubation, stop the reaction with 400 µL of the Stop Solution. Mix well.
- Centrifuge at top speed for 2 minutes in a microcentrifuge.
- Load 200 µL of the supernatant into a plate.
- Read at 525 nm (absorbance) in endpoint mode.
- 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 g-monohydroxamate (Sigma, Catalog # G2253).Per Well:
- rhTGM3: 0.4 µg
- Substrate: 10 mM
Background: Transglutaminase 3/TGM3
Transglutaminase 3 (TG3), also known as epidermal Transglutaminase (Tgase E), belongs to the family of Transglutaminase enzymes that catalyze the posttranslational modification of proteins via calcium dependent cross-linking reactions (1-3). TG3 is involved in the formation of the cornified envelope in skin keratinocytes (4). It functions to cross-link structural proteins during epidermal terminal differentiation. TG3 has been implicated as the dominant autoantigen in dermatitis herpetiformis (5). TG3 activation requires proteolysis of the 77 kDa zymogen into two fragments of approximately 50 and 27 kDa to form the active enzyme (1).
- Kim, I.G. et al. (1993) J. Biol. Chem. 268:12682.
- Griffin, M. et al. (2002) Biochem. J. 368:377.
- Lorand, L. and R.M. Graham (2003) Nat. Rev. Mol. Cell Biol. 4:140.
- Eckert, R.L. et al. (2005) J. Invest. Dermatol. 124:481.
- Sardy, M. et al. (2002) J. Exp. Med. 195:747.
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