Recombinant Human Transglutaminase 1/TGM1 Protein, CF
Recombinant Human Transglutaminase 1/TGM1 Protein, CF Summary
Product Specifications
Met2-Ala817, with an N-terminal Met and 6-His tag
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.
7765-TG
| Formulation | Supplied as a 0.2 μm filtered solution in Tris, NaCl, EDTA, DTT 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
- Assay Buffer: deionized water
- Recombinant Human Transglutaminase 1/TGM1 (rhTGM1) (Catalog # 7765-TG)
- Substrate: Z-Gln-Gly (Sigma, Catalog # C6154), 500 mM, pH 9.0 in deionized water
- Hydroxylamine Hydrochloride (Sigma, Catalog # 159417), 1 M, pH 6.0 in deionized water
- 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 containing 62.5 mM Substrate, 250 mM MES pH 6.0, 12.5 mM DTT, 12.5 mM CaCl2, and 125 mM Hydroxylamine Hydrochloride.
- Dilute rhTGM1 to 0.2 mg/mL in Assay Buffer.
- Mix 30 µL dilute rhTGM1 with 120 µL Substrate Mixture. Include an Enzyme Control containing 30 µL Assay Buffer with 120 µL Substrate Mixture.
- Incubate at 37 °C for 2 hours.
- Stop each reaction with 600 µL of Stop Solution. Mix well.
- Centrifuge at top speed for 2 minutes in a microcentrifuge.
- Load 200 µL of the supernatant into a 96 well clear plate.
- Read plate 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 Enzyme Control
**Derived using calibration standard L-glutamic acid gamma -monohydroxamate (Sigma, Catalog # G2253).
- rhTGM1: 1.6 µg
- Substrate: 10 mM
Reconstitution Calculator
Background: Transglutaminase 1/TGM1
Transglutaminase 1, also known as TGM1, Transglutaminase K, keratinocyte transglutaminase, or epidermal TGase, is a member of the transglutaminase family. Transglutaminases catalyze the calcium-dependent formation of isopeptide bonds between lysine and glutamine residues (1). TGM1 plays an important role in formation of the cornified envelope of differentiating keratinocytes in the epidermis through its cross-linking of epidermal proteins (2). The majority of cases of lamellar ichthyosis result from mutations present within the TGM1 gene and the subsequent dysfunction of TGM1 (1, 3).
- Lorand, L. and R.M. Graham (2003) Nature Rev. Mol. Cell. Biol. 4:140.
- Eckert, R.L. et al. (2005) J. Invest. Dermatol. 124:481.
- Farasat, S. et al. (2009) J. Med. Genet. 46:103.
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