Protocol detail

Western Blot Analysis of Adipose Proteins

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Tissue protein extracts underwent gel electrophoresis and proteins were then transferred to membranes (Arsenijevic et al., 2015 (link)). Membranes were pre-incubated with 1% casein (Vectorlab) for 2 h and then rinsed. Membranes were then incubated 2 h with one of the following primary antibodies—uncoupling protein-1 (UCP1) dilution 1/5000 (cat. no. UCP11, Alpha Diagnostics), SIRT1 dilution 1/200 (sc-19857, Santa Cruz), farnesoid x receptor (FXR) dilution 1/200 (sc-13063, Santa Cruz), and beta-actin dilution 1/1000 (Cat No. 4970–Cell Signaling). Secondary antibody LI-COR anti-rabbit (dilution 1/15000) or anti-goat (dilution 1/15000) were used to detect bands (de Bilbao et al., 2009 (link)). The signals were visualized with the use of Odyssey Infrared Imaging System (Li-Cor Biosciences, Bad Homburg, Germany).

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Arsenijevic D., Cajot J.F., Fellay B., Dulloo A.G., Van Vliet B.N, & Montani J.P. (2016). Uninephrectomy-Induced Lipolysis and Low-Grade Inflammation Are Mimicked by Unilateral Renal Denervation. Frontiers in Physiology, 7, 227.

Publication 2016

casein sc-19857 sc-13063 beta-actin Odyssey Infrared Imaging System

Antibodies Antibody Beta actin Casein Diagnostics Dilution Electrophoresis Goat Membranes Proteins Rabbit Sirt1 Tissue Uncoupling protein 1

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Variable analysis

independent variables

Primary antibodies used (UCP1, SIRT1, FXR, and beta-actin)

dependent variables

Protein expression levels of UCP1, SIRT1, FXR, and beta-actin

control variables

Tissue protein extracts
Gel electrophoresis and protein transfer to membranes
Membrane pre-incubation with 1% casein for 2 hours
Secondary antibodies (anti-rabbit and anti-goat) used for detection
Visualization with Odyssey Infrared Imaging System

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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