Sds page gel

Proteins were either denatured in sample buffer containing beta-mercaptoethanol or nonreducing sample buffer and boiled at 100 °C for 3 min. All samples were separated on standard 10% or gradient 4–20% SDS-PAGE gels (ThermoFisher) and electroblotted to nitrocellulose using an iBlot 2 system. Western blot analysis was performed with antibodies as indicated, followed by incubation with infrared fluorophore-labeled secondary antibodies (Rockland). Bands were detected using a Li-Cor Odyssey infrared scanner.
For cysteine-labeling western blot experiments, thiols were labeled with 10 mM NEM (Sigma) for 3 h RT in the dark or 10uM IRDYE 800CW Maleimide (Li-Cor) for 30 min RT in the dark. Proteins were separated using 4–20% SDS-PAGE gels (ThermoFisher) and IRDYE 800 signal was detected using a Li-Cor infrared Odyssey scanner. NEM labeling was detected using an antibody specific for NEM labeled proteins, OX133 (Absolute Antibody). Relative protein amount was normalized to signals obtained using secondary antibodies against mouse Fc.
NTF challenges were performed by mixing synthetic NTF peptides with recombinant proteins as described in individual experiments; NTF was composed of a 41 amino acid sequence validated by mass spectrometry as described before¹² .

Human fibronectin: 6 µg per lane was cleaved with three different enzymes for 30 min at 37 °C, with the following amounts of enzyme: human tryptase 136 ng, HC 272 ng and the opossum chymase 500 ng. Human fibrinogen: 4 µg per lane was cleaved with three different enzymes for 30 min at 37 °C, with the following amounts of enzyme, human tryptase 136 ng, HC 136 ng, and opossum chymase 500 ng. After incubation, the reactions were stopped with the addition of 3 µL of 4× sample buffer. Half a microliter of β-mercaptoethanol was then added to each sample followed by heating for 7 min at 85 °C. The reaction mixtures were then analyzed on 4–12% precast SDS-PAGE gels (Novex, Invitrogen, Camarillo, CA, USA). To visualize the proteins, the gels were stained overnight in colloidal Coomassie staining solution and destained with 25% (v/v) methanol in ddH₂O for 4 h [47 (link)].

Twenty-five µg of total protein per well was loaded on fixed polyacrylamide concentration of 15 % SDS-PAGE gels (Invitrogen, Thermo Fisher Scientific) and run in MES buffer (Invitrogen, Thermo Fisher Scientific). The total protein content was visualized using the Coomassie protein stain.