Recombinant Mouse Transglutaminase 2/TGM2 Protein, CF Summary
Ala2-Ala686, with an N-terminal Met and 6-His tag
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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 dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.|
|Stability & Storage:||Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
- 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
- Prepare Substrate Mixture by combining right before running assays (Note: Dilute all components to the correct concentration in Assay Diluent.):
- 15 µL of 500 mM Substrate
- 75 µL of 400 mM MES, pH 6.0
- 7.5 µL of 200 mM DTT
- 7.5 µL of 200 mM CaCl2
- 15 µL of 1 M Hydroxylamine Hydrochloride.
- Dilute rmTGM2 to 0.1 mg/mL in Assay Diluent.
- 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.
- Incubate at 37 °C for 2 hours.
- After incubation, stop the reaction with 600 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:
(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).
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).
- rmTGM2: 0.8 µg
- Substrate: 10 mM
Background: Transglutaminase 2/TGM2
Transglutaminase 2 (TG2), encoded by the Tgm2 gene, is also known as tissue Transglutaminase (tTG), Transglutaminase C (TGC), and protein-glutamine-gamma ‑glutamyltransferase. It belongs to the family of transglutaminases that catalyze the posttranslational modification of proteins via calcium dependent cross-linking reactions (1‑3). In addition to its function in protein cross-linking, TGM2 is also capable of hydrolyzing both GTP and ATP (4) and has intrinsic kinase activity (5). TGM2 has been implicated in a variety of human diseases including celiac disease, inclusion body myositis, atherosclerosis, and neurodegenerative diseases (6, 7).
- Gentile, V. et al. (1991) J. Biol. Chem. 266:478.
- Chen, J.S.K. and Mehta K. (1999) Internat. J. Biochem. Cell Biol. 31:817.
- Griffin, M. et al. (2002) Biochem. J. 368:377.
- Lai, T.S. et al. (1998) J. Biol. Chem. 273:1776.
- Mishra, S. et al. (2007) J. Biol. Chem. 282:18108.
- Kim, S-Y. et al. (2002) Neurochem. Int. 40:85.
- Lesort, M. et al. (2000) Prog. Neurobiol. 61:439.
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