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Hybond electrochemiluminescence ecl nitrocellulose membrane

Manufactured by GE Healthcare
Sourced in United Kingdom

Hybond electrochemiluminescence (ECL) nitrocellulose membranes are a type of laboratory equipment used in Western blotting and other protein detection techniques. These membranes provide a surface for the immobilization and detection of proteins that have been separated by electrophoresis. The membranes are made of nitrocellulose and are designed to work with electrochemiluminescence-based detection methods.

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4 protocols using hybond electrochemiluminescence ecl nitrocellulose membrane

1

Molecular Mechanisms of Metabolic Regulation

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Piperine, metformin, Sodium L-lactate, Guanosine 5′-diphosphate sodium salt (GDP, UCP1 inhibitor) and monoclonal anti-β-actin antibody were purchased from Sigma Chemical Company (St. Louis, MO, USA). Compound C (AMPK inhibitor) was provided by Merck (Rahway, NJ, USA). SB203580 (p38 MAPK inhibitor) and monoclonal antibody against UCP1 were purchased from Abcam (Cambridge, MA, USA). Monoclonal antibodies against phosphorylated AMPKα, phosphorylated p38 MAPK, phosphorylated TBC1D4, ACC, p38 MAPK, TBC1D4 and Glut4 and polyclonal antibodies against AMPKα and phosphorylated ACC were purchased from Cell Signaling Technology (Danvers, MA, USA). Monoclonal anti-c-Myc antibody was acquired from Santa Cruz Biotechnology (Dallas, TX, USA). Hybond electrochemiluminescence (ECL) nitrocellulose membranes were obtained from GE Healthcare (Little Chalfont, Buckinghamshire, UK).
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2

AMPK and AKT Signaling Regulation

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BGN was purchased from Flarebio (College Park, MD, USA). STO-609 (an inhibitor of calcium/calmodulin-dependent protein kinase kinase, CAMKK) was purchased from SignalChem (St. Louis, MO, USA). 5-aminoimidazole-4-carboxamide ribonucleotide was purchased from Toronto Research Chemical Incorporation (Toronto, ON, Canada). Polyclonal antibodies against phosphorylated AMPKα, acetyl-CoA carboxylase (ACC), phosphorylated ACC, and β-actin were purchased from Millipore (Billerica, MA, USA). AMPKα, AKT, phosphorylated AKT, forkhead box protein O1 (FoxO1), phosphorylated FoxO1, AKT substrate of 160 kDa (AS160), and phosphorylated AS160 were purchased from Cell Signaling Technology (Beverly, MA, USA). GLUT4 was purchased from Abcam (Cambridge, UK). Compound C (an AMPK inhibitor) was provided by Merck (Rahway, NJ, USA). LY-294002 (an AKT inhibitor), Fluo-3 AM, Ionomycin, forskolin, and Verapamil (a calcium channel blocker) were purchased from Sigma (St. Louis, MO, USA). Hybond electrochemiluminescence (ECL) nitrocellulose membranes were obtained from GE Healthcare (Little Chalfont, Buckinghamshire, UK).
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3

Monitoring SICLOPPS Protein Expression

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SICLOPPS construct expression and processing were monitored by western blot. Cells were recovered from experimentally treated wells and lysed in 1× SDS-PAGE sample buffer. Samples were resolved on a 10% SDS-PAGE gel and transferred onto Hybond electrochemiluminescence (ECL) nitrocellulose membrane (GE Healthcare) by wet transfer overnight at 4°C using a constant voltage of 30 V. SICLOPPS expression and processing products were detected by ECL or quantitative (Li-Cor) western blotting.
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4

Protein Extraction and Western Blot Analysis

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Cells were lysed in buffer containing 50 mM Tris (pH, 7.9), 150 mM NaCl, 1% NP-40, 1 mM EDTA, 10% glycerol, 1 mM sodium vanadate, and protease inhibitor cocktail (Roche, Indianapolis, IN). Proteins were separated via electrophoresis on 4–15% gradient polyacrylamide gels with sodium dodecyl sulfate, transferred to a Hybond electrochemiluminescence (ECL) nitrocellulose membrane (GE Healthcare Biosciences, Piscataway, NJ), and blocked in 5% bovine serum albumin in PBS solution. The membrane was then incubated with primary and secondary antibodies, and target proteins were detected via ECL detection reagent (GE Healthcare Biosciences).
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