Recombinant Human Transglutaminase 7/TGM7 Protein, CF
Recombinant Human Transglutaminase 7/TGM7 Protein, CF Summary
Product Specifications
Asp2-Pro710, with an N-terminal Met and 6-His tag
Accession # Q96PF1
Analysis
Product Datasheets
Carrier Free
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.
5426-TG
| 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 Procedure
- 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
- Prepare substrate mixture right before running assay. Mix the following components per reaction:
- 15 µL 500 mM Z-Gln-Gly
- 75 µL 400 mM MES, pH 6.0
- 7.5 µL 200 mM DTT
- 7.5 µL 200 mM CaCl2
- 15 µL 1 M Hydroxylamine Hydrochloride
- Dilute rhTGM7 to 0.1 mg/mL in deionized water.
- 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.
- 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.
- Transfer 200 µL (in duplicate) 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 assay. (For example: If there are 4 reaction vials, including blanks, multiply all volumes by 5)
|
Specific Activity (pmoles/min/µg) = |
Adjusted Abs* (OD) x Conversion Factor** (pmole/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:- rhTGM7: 0.8 µg
- Substrate: 10 mM
Reconstitution Calculator
Background: Transglutaminase 7/TGM7
Transglutaminase 7 (TG7), encoded by the TGM7 gene, is also known as protein-glutamine-g-glutamyltransferase Z (Tgase Z) (1). It belongs to the family of Transglutaminases that catalyze the posttranslational modification of proteins via calcium dependent cross-linking reactions (2-4). TG7 is ubiquitously expressed in humans (1). Members of the TGM family have been implicated in a variety of human diseases including neurodegenerative diseases, celiac disease, lamellar ichthyosis, bleeding disorders, cataract formation, atherosclerosis, and others (5).
- Grenard, P. et al. (2001) J. Biol. Chem. 276:33066.
- 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.
- Kim, S-Y. et al. (2002) Neurochem. Int. 40:85.
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