Transglutaminase Activity Assay Kit (Colorimetric) (ab204700)
Key features and details
- Assay type: Enzyme activity
- Detection method: Colorimetric
- Platform: Microplate reader
- Assay time: 2 hr 30 min
- Sample type: Adherent cells, Suspension cells, Tissue
- Sensitivity: 10 µU
Overview
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Product name
Transglutaminase Activity Assay Kit (Colorimetric) -
Detection method
Colorimetric -
Sample type
Tissue, Adherent cells, Suspension cells -
Assay type
Enzyme activity -
Sensitivity
10 µU -
Assay time
2h 30m -
Product overview
Transglutaminase Activity Assay Kit (ab204700) utilizes the deamidation reaction of the transglutaminase enzyme with a donor and acceptor substrate resulting in the formation of a hydroxamate product. The hydroxamate product reacts with the Stop Solution forming a purple complex that can be measured colorimetrically at 525 nm. The limit of quantification of this assay is ~10 µU in cell/tissue lysates.
Transglutaminase assay protocol summary:
- add samples and standards to wells
- add reaction mix and incubate for 2 hrs
- add stop solution and centrifuge plate at 1800 g for 15 min
- transfer supernatant to fresh plate
- analyze with microplate reader -
Notes
Transglutaminases (EC 2.3.2.13) are calcium dependent enzymes that catalyze the post-translational modification of proteins by formation of isopeptide bonds. This occurs either through protein cross-linking via formation of γ-glutamyl-ε-lysine bonds or through incorporation of primary amines at selected peptide-bound glutamine residues. The transglutaminase enzyme family comprises the intracellular forms (TG1, TG3 and TG5) expressed mostly in the epithelial tissue; TG2 which is both intracellular and extracellular and expressed in various tissue types; TG4 which is expressed in the prostate gland; factor XIII which is expressed in blood; TG6 and TG7, whose tissue distribution is unknown and band 4.2 (lacking enzymatic activity) which is present on erythrocyte membranes. Transglutaminases also exhibit GTPase, phosphodiesterase and protein kinase activity. Transglutaminases are associated with certain neurological and autoimmune disorders and also cancer.
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Platform
Microplate reader
Properties
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Storage instructions
Store at -20°C. Please refer to protocols. -
Components 100 tests Acceptor Substrate 2 vials Donor Substrate 1 vial DTT 1 x 125µl Homogenization Buffer (10x) 1 x 10ml Hydroxamate Standard 1 vial Plate Sealer 1 unit Positive Control 1 vial Stop Solution 1 x 5ml TG Assay Buffer 1 x 12ml -
Research areas
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Relevance
Transglutaminases (EC 2.3.2.13) are calcium dependent enzymes that catalyze the post-translational modification of proteins by formation of isopeptide bonds. This occurs either through protein cross-linking via formation of ?-glutamyl-e-lysine bonds or through incorporation of primary amines at selected peptide-bound glutamine residues. The transglutaminase enzyme family comprises the intracellular forms (TG1, TG3 and TG5) expressed mostly in the epithelial tissue; TG2 which is both intracellular and extracellular and expressed in various tissue types; TG4 which is expressed in the prostate gland; factor XIII which is expressed in blood; TG6 and TG7, whose tissue distribution is unknown and band 4.2 (lacking enzymatic activity) which is present on erythrocyte membranes. Transglutaminases also exhibit GTPase, phosphodiesterase and protein kinase activity. Transglutaminases are associated with certain neurological and autoimmune disorders and also cancer.
Images
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Transglutaminase Activity Assay Kit (ab204700)
Typical Hydroxamate Standard calibration curve.
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Transglutaminase Activity Assay Kit (ab204700)Transglutaminase activity in HepG2 cells (human hepatoblastoma cell line) and rat liver lysate: HepG2 cells were stimulated with vehicle (DMSO), IL6 (1 µM), Dexamethasone (DXM -1 µM), or with IL6 (1µM) and DXM (1 µM). Approximately 250 µg protein was used for determining transglutaminase activity in cells and tissue lysate. Activity is expressed as nmoles of product formed in 2h and is normalized to the protein amount.
Note: HepG2 cells have similar intrinsic Transglutaminase activity in the presence or absence of vehicle control (DMSO).
