Ppsq 51a 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).

The osteosarcoma cell line MG-63 was grown in Eagle's minimum essential medium and stimulated with the synthetic double stranded RNA poly rI:rC (50 μg/ml; P-L Biochemicals, Milwaukee, WI) to produce CXCL8 as described (29 (link)). To isolate CXCL8 variants, MG-63 cell culture supernatant was concentrated and partially purified using controlled pore glass (CPG) and heparin affinity chromatography (GE Healthcare; Chicago, IL). Protein elution was achieved using a NaCl gradient of 0.05–2.0 M in 50 mM Tris-HCl (pH 7.4). Heparin-Sepharose fractions containing CXCL8 immunoreactivity, demonstrated by a specific CXCL8 ELISA developed in our laboratory (20 (link)), were further purified on a C8 Aquapore RP-300 column (220 ×2.1 mm, PerkinElmer Life Sciences; Waltham, MA) by RP-HPLC (Waters 600 HPLC System: controller and solvent delivery system; Milford, MS). CXCL8 elution was achieved through an acetonitrile gradient (0–80%) in 0.1% (v/v) TFA/ultrapure water (pH 2.0). UV absorbance was measured at 214 nm reflecting protein concentrations. CXCL8 proteoform identification was performed by Edman degradation on the purified fractions using a PPSQ-51A protein sequencer (Shimadzu, Kioto, Japan) and via nano-LC-MS/MS.