Ez link pentylamine biotin

Recombinant murine thrombin (Haematologic Technologies Inc) was reconstituted to 250 U/mL in ddH₂O and stored at −80 °C. Recombinant human tPA (Pathway Diagnostics) was diluted in Tris-buffered saline (TBS; 0.05 M Tris HCl, 0.1 M NaCl, pH 7.4) to 1,400 nM and stored at −80 °C. EZ-link pentylamine-biotin (Thermo Fisher Scientific) was diluted in ddH₂O to 30 μM and stored at −20 °C. AlexaFluor⁴⁸⁸, AlexaFluor⁶⁴⁷, and AlexaFluor⁶⁸⁰ protein labeling kits were purchased from Thermo Fisher Scientific. All other chemicals were obtained from Sigma unless stated otherwise.

ELISA wells were coated O/N at 4 °C for 16 h with 120 µL of 15 µg/mL N,N′-dimethyl casein (DMC) in PBS. After one wash with PBS, 100 µL of reaction mix (100 mM Tris-HCl, pH 8.0, 0.2 mM EZ-Link™ Pentylamine-Biotin from Thermo Scientific cat#21345, 10 mM DTT, 5 mM CaCl₂) were added according to the test: (A) with increasing amounts of recombinant TG2; (B) with 50 ng of recombinant TG2 and increasing concentrations of CaCl₂; (C) with 50 ng of recombinant TG2 in the presence or in the absence of 100 µM guanosine 5′- triphosphate (GTP); (D) with 50 ng of recombinant TG2 in the presence or in the absence of hTG2-specific peptide inhibitors 1-155, R281 or ZDON (Sigma-Aldrich, cat#616467) at three different concentrations (25 mM, 2.5 mM and 0.25 mM). Samples were added in each well and the plate was incubated for 1 h at 37 °C. Following three washes with PBS-Tween 20 0.1% and three with PBS, the plate was incubated for 1 h at 37 °C with 100 µL per well of Pierce™ High Sensitivity Streptavidin-HRP (Thermo Scientific, cat#21134) diluted in a solution of 1% BSA in PBS in volumetric ratio 1:200. After washes, the colorimetric reaction was developed by adding 100 µL per well of TMB and stopped after 5 min by adding 50 µL per well of 2.5 mM H₂SO₄. The absorbance was measured at 450 nm using a microplate reader.

ELISA wells were coated O/N at 4 °C for 16 h with 120 µL of 15 µg/mL N,N′-dimethyl casein (DMC) in PBS. After one wash with PBS, 100 µL of reaction mix (100 mM Tris-HCl, pH 8.0, 0.2 mM EZ-Link™ Pentylamine-Biotin from Thermo Scientific cat#21345, 10 mM DTT, 5 mM CaCl₂) with HEK293 cell protein lysates were added in each well and the plate was incubated for 1 h at 37 °C. For protein lysates of HEK293 cells after transient transfection, 1 μg of lysate per well was used. For protein lysates of hygromycin-selected HEK293 stable clones expressing TGs2, the quantity of samples used was normalized on the level of TG2 expression, measured by quantifying the relative protein band on the nitrocellulose membrane with Image Lab Software version 6.1 (Bio-Rad). Following three washes with PBS-Tween 20 0.1% and three with PBS, the plate was incubated for 1 h at 37 °C with 100 µL per well of Pierce™ High Sensitivity Streptavidin-HRP diluted in a solution of 1% BSA in PBS in volumetric ratio 1:200. After washes, the colorimetric reaction was developed by adding 100 µL per well of TMB and stopped after 5 min by adding 50 µL per well of 2.5 mM H₂SO₄. The absorbance was measured at 450.

Zebrafish embryos protein lysates were obtained starting from 30 embryos at 1, 3, and 5 dpf. The embryos were lysed first by pipetting with an insulin syringe in 600 μL of lysis buffer (50 mM Tris-HCl, pH8, 150 mM NaCl, 1% Triton X100), and then by sonication. After 15 min of centrifugation at 14,000× g at 4 °C, the clear lysates were quantified with Bradford reagent. ELISA wells were coated O/N at 4 °C for 16 h with 120 µL of 15 µg/mL N,N′-dimethyl casein (DMC) in PBS. After one wash with PBS, 100 µL of reaction mix (100 mM Tris-HCl, pH 8.0, 0.2 mM EZ-Link™ Pentylamine-Biotin from Thermo Scientific cat#21345, 10 mM DTT, 5 mM CaCl₂) with 20 μg of zebrafish embryos protein lysates were added in each well and the plate was incubated for 2 h at 37 °C. Following three washes with PBS-Tween 20 0.1% and three with PBS, the plate was incubated for 1 h at 37 °C with 100 µL per well of Pierce™ High Sensitivity Streptavidin-HRP diluted in a solution of 1% BSA in PBS in volumetric ratio 1:200. After washes, the colorimetric reaction was developed by adding 100 µL per well of TMB and stopped after 5 min by adding 50 µL per well of 2.5 mM H₂SO₄. The absorbance was measured at 450 nm using a microplate reader (TECAN^®, Männedorf, Switzerland).