Western Blot Analysis of Liver Proteins

Liver sonicates were obtained as described previously (Fernandes et al., 2018 (link)), mixed with 4X sample loading buffer (Invitrogen), and 20–50 ug protein fractionated in 4–12% NuPAGE BisTris gels (Life Technologies). Samples were transferred to Immobilon-FL PVDF transfer membrane (Millipore) and probed overnight with antibodies, as indicated, at a 1:2,500 dilution. Fluorescent-labeled secondary antibodies (LI-COR Biosciences) were used at 1:2,500 for 1 hr. Blots were imaged and quantified using the LI-COR Odyssey instrument.

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Fonseca T.L., Fernandes G.W., Bocco B.M., Keshavarzian A., Jakate S., Donohue TM J.r., Gereben B, & Bianco A.C. (2019). Hepatic inactivation of the type 2 deiodinase confers resistance to alcoholic liver steatosis. Alcoholism, clinical and experimental research, 43(7), 1376-1383.

Publication 2019

4X sample loading buffer NuPAGE BisTris gels Immobilon-FL PVDF transfer membrane Fluorescent-labeled secondary antibodies

Antibodies Bistris Buffer Dilution Fluorescent antibodies Gels Immobilon Liver Membrane Protein Pvdf

Corresponding Organization : University of Chicago

Other organizations : Rush University, University of Nebraska Medical Center, Hungarian Academy of Sciences, HUN-REN Institute of Experimental Medicine

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Variable analysis

independent variables

Liver sonicates

dependent variables

Protein fractionation
Antibody binding
Fluorescent signal intensity

control variables

Sample loading buffer (4X)
Protein amount (20-50 μg)
Gel type (4-12% NuPAGE BisTris gels)
Transfer membrane (Immobilon-FL PVDF)
Antibody dilution (1:2,500)
Secondary antibody dilution (1:2,500)
Imaging and quantification (LI-COR Odyssey instrument)

controls

Positive control: Not specified
Negative control: Not specified

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