Transglutaminase, Streptoverticillium mobaraense (TG) is an enzyme that forms cross-links between protein molecules. Transglutaminase, Streptoverticillium mobaraense attaches proteins and peptides to small molecules, polymers, surfaces, DNA and other proteins. Transglutaminase, Streptoverticillium mobaraense is widely used in food applications in the meat, fish, dairy and baking industries^[1][2][3].

Tissue Transglutaminase is used to detect T cell proliferation experiments^[4]
(1) Culture PBMC cells in a 96-well plate using complete RPMI and heat-inactivated 10% human AB serum, with 1×10⁵ cells per well. Add 12 μg/mL of peptide, 50 μg/mL of trypsin-digested gliadin, or phosphate-buffered saline, along with Tissue Transglutaminase. The total volume per well is 200 μL, and incubate at 37°C, 5% CO₂, and humid air for 5 days.
(2) Add 1 microcurie of ³H-thymidine to each well. After continuing the incubation for another 18 hours, measure the incorporation of ³H in DNA using a cell harvester and a radioactive counter. The results are expressed as counts per minute (c.p.m.) for the test samples.
Tissue Transglutaminase is used in HLA class II restriction experiments^[4]
(1) Gliadin (100 mg/ml) is digested with α-chymotrypsin (weight ratio 200:1) at room temperature in 0.1 M ammonium bicarbonate and 2 M urea.
(2) After the digestion process lasts for 24 hours, terminate the reaction by heating the samples to 98°C for 10 minutes.
(3) After centrifuging the samples at 13,000 g for 10 minutes, collect the supernatant and sterile filter it using a 0.2-micron filter membrane.
(4) Verify the digestion of gliadin by SDS-PAGE and determine the protein concentration.
(5) Treat gliadin (640 μg/mL) and synthetic gliadin peptide segments (15 amino acid segments: 160 μg/mL, other segments: 0.1 mM) with the products of α-chymotrypsin digestion at 37°C using 50 μg/mL of Tissue Transglutaminase in PBS buffer containing 1 mM CaCl₂ for 2 hours.
(6) The treated peptide segments and peptide mixtures are aliquoted into sterile 96-well plates and stored frozen at -20°C.
(7) Wash the anti-CD4 or CD8 antibody beads four times with RPMI medium, and incubate them on ice with 5×10⁶ PBMC cells for 30 minutes.
(8) Remove the magnetic beads and perform a cell count.
(9) Pre-incubate 5×10⁶ PBMC cells with monoclonal antibodies against HLA-DR (L243), HLA-DQ (L2), and HLA-DP (B7.21) (10 μg/mL each) at room temperature for 1 hour.
(10) Then add the peptide segments and use the ELISPOT method to detect the secretion of IFN-γ, assessing T cell response to the peptide segments.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Kieliszek M, Misiewicz A. Microbial transglutaminase and its application in the food industry. A review. Folia Microbiol (Praha). 2014 May;59(3):241-50.  [Content Brief]

  [2]. Pavel Strop, et al. Versatility of microbial transglutaminase. Bioconjug Chem. 2014 May 21;25(5):855-62.  [Content Brief]

  [3]. Anna Bérot, et al. Generation of Anti-Epidermal Growth Factor Receptor‑2 (HER2).ACS Pharmacology & Translational Science.2024 American Chemical Society

  [4]. R P Anderson, et al. In vivo antigen challenge in celiac disease identifies a single transglutaminase-modified peptide as the dominant A-gliadin T-cell epitope. Nat Med. 2000 Mar;6(3):337-42.  [Content Brief]