Western blot analysis was conducted as previously described (Timper et al., 2017 (link)). In brief, membranes were blocked with 10% WB-blocking reagent (Roche, Switzerland) in Tris-buffered saline containing 0.1% Tween-20 (TBS-T), and incubated with primary antibodies, made up in TBS-T (0.1% Tween-20) and 5% Western Blotting-Reagent, at 4°C overnight. The following primary antibodies and dilutions were used: anti-GLP-1R (1:1000, #NBP1-97308, Novus Biologicals), anti-Akt (#4686, Cell Signaling), anti-p-Akt(S473) (#4060, Cell Signaling), anti-NDUFA9 (1:1000, #459100, Invitrogen), anti-NDUFB8 (1:1000, #459210, Invitrogen), anti-Complex II 70 kDa Fp Subunit (1:1000, #459200, Invitrogen), anti-UQCRC1 (1:1000, #459140, Invitrogen), anti-Complex IV (1:1000, #459110, Invitrogen), anti-ATP synthase subunit β (1:1000, #21351, Invitrogen), anti-p-Ser-226-GLUT-1 (#ABN991, Merck Millipore), anti-GLUT-1 (#07-1401, Merck Millipore), anti-FGF21 (#AF3057, R&D Systems) and, as loading control: anti-calnexin (1:4000, #208880, Merck Milipore). Quantification of chemiluminescent signals was performed with the ImageJ (Fiji)-software package (Schneider et al., 2012 (link)).
Anti fgf21
Manufactured by R&D Systems
Anti-FGF21 is a protein that binds to and neutralizes the activity of Fibroblast Growth Factor 21 (FGF21). FGF21 is a metabolic regulator that plays a role in glucose and lipid homeostasis. Anti-FGF21 can be used to study the biological functions of FGF21.
Automatically generated - may contain errors
Lab products found in correlation
Anti pakt s473, by Cell Signaling Technology (1 mentions)
Anti calnexin, by Merck Group (1 mentions)
Anti glut1, by Merck Group (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti uqcrc1, by Thermo Fisher Scientific (1 mentions)
Wb blocking reagent, by Roche (1 mentions)
Anti glp 1r, by Novus Biologicals (1 mentions)
Anti ndufa9, by Thermo Fisher Scientific (1 mentions)
Anti ndufb8, by Thermo Fisher Scientific (1 mentions)
Anti complex 2 70 kda fp subunit, by Thermo Fisher Scientific (1 mentions)
Anti complex 4, by Thermo Fisher Scientific (1 mentions)
Iblot2, by Thermo Fisher Scientific (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
Af3057, by R&D Systems (1 mentions)
Anti ucp1 ab10983, by Abcam (1 mentions)
Bca protein assay, by Thermo Fisher Scientific (1 mentions)
2 protocols using anti fgf21
1
Western Blotting Procedure for Protein Analysis
2
Adipose Tissue Protein Extraction Protocol
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White adipose tissue was homogenized in lysis buffer (50 mM HEPES, PH 7.4, 150 mM NaCl, 1 % Triton X-100, 5 mM EGTA) and homogenates were spun at 20,000 g. Supernatants were extracted with 4 volumes of cold acetone and the precipitated protein was resuspended in 10 % SDS, made up to a final SDS concentration of 2 % with water, and spun at 12,000 g for 2 min to obtain the final supernatant. Protein was determined by the BCA protein assay (Thermo Fisher, Rockford, IL). Proteins were separated in 10 % Bis-Tris NuPAGE gels and transferred to PVDF membranes employing the iBlot2 (Thermo Fisher, Rockford, IL). Membranes were blocked and incubated with primary and secondary antibodies in Odyssey blocking buffer (Li-Cor, Lincoln, NE). Primary antibodies were: anti-FGF21, AF3057, and anti-CD137, AF937 (R&D Systems, Minneapolis, MN), anti-P2RX5, sc-398682 (Santa Cruz Biotechnology, Dallas, TX), anti-beta-actin, sc-47778 (Santa Cruz, Dallas, TX), and anti-UCP1, ab10983, (abcam, Cambridge, MA). Dye-conjugated secondary antibodies were from Li-Cor (Lincoln, NE). The bands were detected by immunofluorescence.
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