Recombinant Mouse Transglutaminase 2/TGM2 Protein, CF Summary
Details of Functionality |
Measured by its ability to form CBZ-Gln-Gly-Hydroxamate from CBZ-Gln-Gly and Hydroxylamine. The specific activity is >1,300 pmol/min/μg, as measured under the described conditions. |
Source |
Spodoptera frugiperda, Sf 21 (baculovirus)-derived mouse Transglutaminase 2/TGM2 protein Ala2-Ala686, with an N-terminal Met and 6-His tag |
Accession # |
|
N-terminal Sequence |
Met |
Protein/Peptide Type |
Recombinant Enzymes |
Gene |
Tgm2 |
Purity |
>90%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining. |
Endotoxin Note |
<1.0 EU per 1 μg of the protein by the LAL method. |
Applications/Dilutions
Dilutions |
|
Theoretical MW |
78 kDa. Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors. |
SDS-PAGE |
78 kDa, reducing conditions |
Packaging, Storage & Formulations
Storage |
Use a manual defrost freezer and avoid repeated freeze-thaw cycles.- 6 months from date of receipt, -70 °C as supplied.
- 3 months, -70 °C under sterile conditions after opening.
|
Buffer |
Supplied as a 0.2 μm filtered solution in Tris, NaCl and Glycerol. |
Purity |
>90%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining. |
Assay Procedure |
- 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.
(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 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:
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). Per Well:
- rmTGM2: 0.8 µg
- Substrate: 10 mM
|
Notes
This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.
Alternate Names for Recombinant Mouse Transglutaminase 2/TGM2 Protein, CF
Background
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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