Transglutaminase, Streptoverticillium mobaraense

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.