Brain tissues were homogenized in an ice-cold immunoprecipitation buffer (IP buffer) (containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM EDTA, 10% glycerol, 1% Nonidet p-40, phosphatase inhibitor cocktail 2 (Sigma) and protease inhibitor cocktail (Merck)). The homogenate was centrifuged at 15,000g for 30 min at 4 °C and the supernatant was determined using a coomassie protein assay reagent (Thermo). Equal amounts of extracted proteins were respectively loaded on 4–12% NuPAGE Bis-Tris Gels (Thermo) and subjected to immunoblotting. The following primary antibodies were used: rabbit anti-ZO1 (1:500, Thermo); mouse anti-occludin (1:2000, Thermo); rabbit PKC-δ (1:500, cell signaling); mouse anti-GAPDH (1:5000, Proteintech). Appropriate secondary antibodies (GE Healthcare) were incubated with the membranes for 1 h at room temperature. For Co-Immunoprecipitation assay, immunoprecipitated proteins were detected using VeriBlot for IP Detection Reagents (Abcam) without being contaminated by IgG heavy and light chains from the precipitated-antibodies. The signals were visualized using Amersham™ ECL Select Western Blotting Detection Reagent (GE Healthcare) according to the manufacturer’s instructions and recorded via UVP BioSpectrum Image system (UVP). Data were analyzed using Vision Works LS software (UVP).
Uvp biospectrum image system
Manufactured by Analytik Jena
Sourced in United Kingdom
The UVP BioSpectrum Image system is a comprehensive imaging platform designed for the detection and analysis of chemiluminescent, fluorescent, and colorimetric signals. It features a high-resolution CCD camera, motorized filter wheel, and advanced software for image capture and processing.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti zo 1, by Thermo Fisher Scientific (2 mentions)
Coomassie protein assay reagent, by Thermo Fisher Scientific (1 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (1 mentions)
Amersham ecl select western blotting detection reagent, by GE Healthcare (1 mentions)
Mouse anti occludin, by Thermo Fisher Scientific (1 mentions)
Appropriate secondary antibodies, by GE Healthcare (1 mentions)
Veriblot for ip detection reagent, by Abcam (1 mentions)
Visionworks ls software, by Analytik Jena (1 mentions)
Phosphatase inhibitor cocktail 2, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Mouse anti gapdh, by Proteintech (1 mentions)
Anti pparα, by GeneTex (1 mentions)
Anti phospho akt ser473, by Cell Signaling Technology (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Protein assay dye reagent, by Bio-Rad (1 mentions)
Anti β actin, by GeneTex (1 mentions)
Anti pi3 kinase p85, by Cell Signaling Technology (1 mentions)
Anti insulin receptor β, by Cell Signaling Technology (1 mentions)
2 protocols using uvp biospectrum image system
1
Analysing Brain Tissue Protein Expression
2
Western Blot Protein Analysis Protocol
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The protein extraction methods used in this study were adopted from our previous protocol [11 (link)]. The protein concentration in the cell extract was determined using a Bio-Rad protein assay dye reagent (Richmond, VA, USA). Aliquots of the supernatant containing 50 μg protein were separated through standard SDS-PAGE and electrophoretically transferred to polyvinylidene difluoride membranes. The membranes were incubated with anti-insulin receptor β (IRβ; 1 : 1000; Cell Signaling Technology, Beverly, MA, USA), anti-PI3-kinase p85 (1 : 1000; Cell Signaling Technology), anti-phospho-Akt (Ser473) (1 : 1000; Cell Signaling Technology), anti-Akt (1 : 500; Cell Signaling Technology, Danvers, MA, USA), antiglucose transporter (GLUT)2 (1 : 500; Millipore, Billerica, MA, USA), anti-PPARα (1 : 1000; GeneTex, Irvine, CA, USA), or anti β-actin (1 : 4000; GeneTex) antibodies at 4°C overnight. The membranes were incubated with anti-mouse IgG or anti-rabbit IgG secondary antibodies and washed thrice for 5 min each time. Protein band images were detected and captured using the UVP Biospectrum image system (Level, Cambridge, UK). Finally, all relevant protein expressions were normalized with β-actin.
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