Cells were lysed in 50 mM Tris-HCl (pH 7.5), 50 mM NaCl, 5 mM MgCl2, 0.1% Triton X-100, and protease inhibitor (cOmplete; Roche). 20 µg of total protein extracts were separated by SDS-PAGE and transferred onto a nitrocellulose membrane (Optitran BA-S 85 Reinforced NC; Whatman). The membrane was incubated in PBS supplemented with 0.4% Tween-20 and 5% dry milk for 1 h. Primary antibodies recognizing Jak1 (6G4; Cell Signaling Technology), Jak2 (D2E12; Cell Signaling Technology), Stat1 (polyclonal; Cell Signaling Technology), Phospho-Stat1 (58D6 [Tyr701]; Cell Signaling Technology), ERAP1 (6H9), MECL (K6512), LMP2 (polyclonal; Abcam), LMP7 (polyclonal; Abcam), or β-Actin (C4; Santa Cruz Biotechnology, Inc.) were diluted in PBS/0.1% Tween-20 and 2.5% dry milk and were applied overnight at 4°C. The membrane was washed three times with PBS/0.1% Tween-20 followed by 1-h incubation with the secondary Ab (0.2 µg/ml goat anti–rabbit IgG H&L chain–specific peroxidase conjugate, rabbit anti–mouse IgG H&L chain–specific peroxidase conjugate, or rabbit anti–goat IgG H&L chain–specific peroxidase conjugate; EMD Millipore). The ECL Prime Western Blotting Detection kit (GE Healthcare) was used for detection. Membranes were analyzed with the Fusion FX System using FusionCapt Advance FX7 Software (Peqlab).
Fusion fx system
Manufactured by Avantor
The Fusion FX System is a lab equipment product designed for spectroscopic analysis. It provides core functionality for sample preparation and data acquisition, without interpretation or extrapolation on intended use.
Automatically generated - may contain errors
Lab products found in correlation
β actin c4, by Santa Cruz Biotechnology (1 mentions)
Jak2 d2e12, by Cell Signaling Technology (1 mentions)
Jak1 6g4, by Cell Signaling Technology (1 mentions)
Stat1, by Cell Signaling Technology (1 mentions)
Ecl prime western blotting detection kit, by GE Healthcare (1 mentions)
Whatman, by Cytiva (1 mentions)
2 protocols using fusion fx system
1
Western Blotting of Immune Regulators
2
Protein Expression Analysis by Western Blot
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Cells were harvested by scraping, lysed in lysis buffer (1% Triton X-100, 10 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM MgCl2 and 5 mM EDTA, pH 8.0) and relative protein content was assayed using the BCA protein assay (Pierce). Equal amounts of protein (30 µg) were separated in an SDS-polyacrylamide gel and transferred to nitrocellulose membrane using semi-dry blotting. Membranes were blocked in PBS/0.05% Tween20 (PBS-T) containing 5% BSA and incubated with primary antibody (0.2 μg/mL) in PBS-T/5% BSA at 4 °C over night. After washing thrice membranes were incubated with species specific horseradish peroxidase coupled antibody (Jackson Laboratories) in PBS-T/5% BSA for 2 h with shaking, washed in PBS-T and specific bands were visualized by enhanced chemiluminescence. Pictures were taken using the Fusion FX System (PeqLab) until sufficient pixel saturation was achieved or using x-ray films.
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