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T054 transglutaminase microassay kit, colorimetric r.n.1.2

Kit produced and Developed by CovalAb, France

INTENDED FOR RESEARCH USE ONLY, NOT FOR USE IN HUMAN, THERAPEUTIC OR DIAGNOSTIC APPLICATIONS. ZEDIRA GmbH, Roesslerstr. 83, D-64293 Darmstadt, Germany; +49 (0)6151-325100 Product No.: T054
Transglutaminases (EC. 2.3.213, R-glutamyl-peptide: amine γ-glutamyltransferase) are a
family of calcium dependent enzymes which catalyse an acyl transfer reaction between the
γ-carboxamide group of peptide bound glutamine and various primary amines.
The ε-amino group of lysine residues as well as some polyamines are the physiological
amine donors, but some non-physiological amines can also be used by the enzyme. In
vertebrates, transglutaminases (TGases) are widely distributed in various organs, tissues
and body fluids. TGases are involved in a variety of roles including blood clotting, formation
of the cornified envelope of the epidermis and its appendages (hair, nail, callus), stabilization
of intra and extra cellular matrices and cross-linking of cell envelopes in apoptosis.
All the enzymatic assays for TGases referred to above were carried out under optimal
conditions of pH, Ca2+ and substrates with dimethylcasein as the first substrate and
radiolabelled putrescine or dansyl cadaverine as the second substrate. Another approach to
quantitating TGase activity has been to measure ammonia released during isopeptide bond
formation. These techniques are time consuming and labor intensive.
To completely overcome these problem we have developed a three labor step solid phase
micro assay: “TG-Covtest”

Principle of the assay

The TG-Covtest uses CBZ-Gln-Gly as the first substrate (amine acceptor) and biotin
cadaverine as second (amine donor). In the 1st step, samples suspected of containing
TGase are incubated with calcium, dithiothreitol (DTT) and biotinylated cadaverine in the
wells of microtiter plates to which CBZ-Gln-Gly had been covalently coupled previously. In
the presence of TGase biotinylated cadaverine is incorporated γ-carboamyde of the
glutaminyl residue of the dipeptide to form γ-glutamyl cadaverine biotin. In the second step
streptavidin-labelled peroxidase is added to the wells. In the third step, peroxidase activity is
revealed using H2O2 as HRP substrate and tetramethyl benzidine as electron acceptor
The TG-Covtest has two original features:
The use of covalently coupled CBZ-Gln-Gly solves the problem of leaching out of absorbed substrate The second feature is that 96 samples can be simultaneously screened in 30 minutes time.
Other supplies required

Microplate reader capable of measuring absorbance at 450 nm.
Pipettes, multi-channel pipette, and pipette tips.
Ultra-pure or deionized water
Squirt bottle, manifold dispenser, or automated microplate washer.
ZEDIRA GmbH, Roesslerstr. 83, D-64293 Darmstadt, Germany; +49 (0)6151-325100 Product No.: T054
Reagents of the Kit

R1: Microtiter plates with covalently bound CBZ-Gln-Gly R3: Biotin-cadaverine/CaCl2 (lyophilized powder)
The Kit contains also recombinant tissue transglutaminase (Zedira, T058) for calibration
Storage and Preparation of Reagents

R1: Microtiter plates with covalently bound CBZ-Gln-Gly
The plates should be kept at +4°C and are ready to use.
R2: Negative control
The negative control should be kept at +4°C and is ready to use.
The negative control contains EDTA which inhibits the TGase activity.
R3: Biotin-cadaverine/CaCl2
Lyophilized powder should be kept at -20°C.
Lyophilised powder should be reconstituted with 6,5 mL of distilled water just before use.
Unused solution should not be stored more than 4 hours at +4°C.
R4: Enzyme tracer
Enzyme tracer should be kept at –20°C.
Enzyme tracer should be diluted at 1/2000 with 1X diluent buffer just before use. Unused
solution should be eliminated
R5: Diluent buffer 10X
The diluent buffer 10X should be kept at +4°C. Dilu ent buffer 10X should be diluted at 1/10
with distilled water just before use.
R6: HRP substrate / R7: Chromogen
The reagents should be kept at +4°C. 1 drop of chro mogen should be added to 2 mL of HRP
substrate just before use.
R8: Blocking reagent
Blocking reagent should be kept at +4°C and is read y to use.

ZEDIRA GmbH, Roesslerstr. 83, D-64293 Darmstadt, Germany; +49 (0)6151-325100 Product No.: T054
Assay procedure

Preliminary Operations
1. Identify a sufficient number of wells to run in duplicate: b) the positive control (TGase + distilled water) c) the negative control (TGase + R2 reagent) Controls and samples should all be subjected to exactly the same assay procedure. Keep one blank well for HRP substrate/chromogen solution only. 2. Dispense 150 µL per well of 1x diluent buffer and incubate the plate for at 3. Prepare the samples: samples from cells or tissues should be centrifuged Perform the TG-Covtest as follows
Remove the 1X diluent buffer from the plate. Per well, dispense the samples* (50 µL), dispense the positive control (40 µL TGase +10 µL distilled water) and dispense the negative control Dispense 50 µL per well of reconstituted biotin-cadaverine/CaCI2 Prepare the enzyme tracer solution at 1/2000. Wash the wells three times with Phosphate Buffered Saline containing 0,1% Tween 20 or Tris Buffered Saline containing 0,1% Tween 20. Dispense 100 µL per well of 1/2000 diluted enzyme tracer solution. Prepare the HRP substrate/chromogen solution. Wash the wells three times with Phosphate Buffered Saline containing 0,1% Tween 20 or Tris Buffered Saline containing 0,1% Tween 20. Dispense 100 µL per well of HRP substrate/chromogen solution. Incubate for 2 to 10 min at room temperature. Dispense 50 µL per well of blocking reagent. Measure the optical density of each well at 450nm (OD450nm). *Please note that it could be better to treat the samples as controls: - active TGase sample: (40 µL sample + 10 µL distilled water) per well, - inactive TGase sample: (40 µL sample + 10 µL R2 reagent) per well. ZEDIRA GmbH, Roesslerstr. 83, D-64293 Darmstadt, Germany; +49 (0)6151-325100 Product No.: T054
Calculation of Results
Several experiments using purified guinea pig TGase (Activity: 2 units per mg protein) have
shown that 1.25 mU/mL (final concentration) correspond to an absorbance value of 1+ 0.05
OD at 450 nm. 1 unit will catalyze the formation of 1µmole of hydroxamate at pH 6.0 at 37°C,
using L-glutamic acid γ-monohydroxamate as the standard (Folk and Cole, 1966). If other
TGase standards are used in this assay the reference value may be different. End user
should establish their own set of reference values.
Safety Procedures
The product is not licensed or approved for administration to humans or to animals.
Standard Laboratory Practices should be followed when handling this material.
Handle with care HRP substrate (R6), chromogen (R7) and blocking reagent (R8).
R6, R7 and R8 reagents are irritating to eyes and skin. In case of contact with eyes,
rinse immediately with plenty of water and seek medical advice.
Comparison of colorimetric
and radiometric FXIII assays
ZEDIRA GmbH, Roesslerstr. 83, D-64293 Darmstadt, Germany; +49 (0)6151-325100 Product No.: T054

Folk JE and Finlayson JS. The  -glutamyl)lysine cross link and the catalytic role of transglutaminases. Advances in Protein Chemistry, 1977, 31: 1-33. El Alaoui S. and al. Transglutaminase activity and N  -glutamyl)lysine isopeptides levels in cells during growth in culture, an enzymatic and immunological study.Int. J. Cancer, 1992, 48,221-226. El Alaoui S. and al. Evaluation of a novel colorimetric solid phase micro-assay for plasma Factor XIII. Blood Coagulation and Fibrinolysis, 199, 3, 803-811 Jeon, W.M. et al. Colorimetric assay for cellular transglutaminase. Anal. Lorand L and al, A filter paper assay for transamidating enzymes using radioactive amine substrate. Anal . Biochem., 1972; 50, 623-631. Thomas F. and al. A microtiter plate transglutaminase Assay utilizing 5-(Biotinamido)pentylamine as substrate, Anal. Biochem., 1992, 205, 166-171. Villalobos E. and al. Molecular cloning and characterization of a maize transglutaminase complementary DNA. Gene 336., 2004, 93-104. Thomas V. and al. Development and evaluation of a modified colorimetric solid-phase microassay for measuring the activity of cellular and plasma (Factor XIII) transglutaminases Biotechnol. Appl. Biochem. (2006) 43, 171– ZEDIRA GmbH, Roesslerstr. 83, D-64293 Darmstadt, Germany; +49 (0)6151-325100 Product No.: T054


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