Procise 491clc protein sequencer

IL-7 samples were separated in Novex 16% tricine gels (Invitrogen, cat. no. EC6695BOX) as recommended by the supplier. Proteins in gels were stained with the SilverQuest Silver Staining Kit (Invitrogen cat. no. LC6070) or transferred onto PVDF membranes using the Trans-Blot Turbo Transfer System with associated materials and protocols (Biorad, cat. no. 1704150). For Edman sequencing, proteins on the PVDF membrane were stained with Coomassie Brilliant Blue (Coomassie R-250, Thermo Scientific, cat. no. 20278) and analyzed with the Procise 491cLC protein sequencer (Applied Biosystems) or the PPSQ-51A protein sequencer (Shimadzu). For the analysis of signaling molecules, proteins in cell lysates were separated in 4-12% Tris-glycine gels under reducing conditions and transferred to PVDF membranes. Membranes were blocked for 1 h in 5% BSA with TBST buffer (150 mM NaCl, 0.1% Tween 20, 50 mM Tris, pH 7.5) and incubated overnight with anti-pSTAT3 (Tyr⁷⁰⁵) (Cell Signaling, cat. no. 9138) or anti-β-actin (Proteintech, cat. no. 20536-1-AP) antibodies. After washing, the blot was incubated with peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG for 1 h at room temperature. Finally, Western blot images were developed using the Vilber Lourmat Fusion system (Labtech International) and Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific).

SDS-PAGE-separated proteins were blotted onto a PVDF membrane (using a BioRad Trans-Blot Turbo RTA Transfer Kit) and stained with Coomassie blue. The bands were cut from the stained membrane, destained in methanol, rinsed with ultrapure water, and then subjected to automated NH₂-terminal amino acid sequence analysis (Procise 491 cLC protein sequencer, Applied Biosystems, Foster City, CA) based on the Edman degradation reaction (Loos et al., 2009 ).

Purified Md1RIP and Md2RIP were analyzed by SDS-PAGE, electroblotted onto a Problot™ polyvinylidene fluoride (PVDF) membrane (Applied Biosystems, Foster City, CA, USA) and the blot stained using 1:1 mix of Coomassie Brilliant Blue and methanol to visualize the protein. The bands of interest were cut and used for N-terminal sequencing by Edman degradation using a capillary Procise 491cLC protein sequencer without alkylation of cysteines (Applied Biosystems).