Western Blot Analysis of Peripheral Nerve Proteins

Peripheral nerves dissected after transcardial perfusion of deeply anesthetized mice with 0.1 M PBS were homogenized in T-PER (Thermo Scientific) and run on Mini-Protean TGX Gels (10%; Bio-Rad) and transferred to nitrocellulose membranes (Bio-Rad). For all Western blots, 15–30 μg of proteins were separated on the gel. Membranes were incubated overnight with MCT1 (generated for laboratory (Morrison et al., 2015 (link)); 1:200), P0 (Aves Labs. Inc.; catalog # PZO; myelin protein-zero chicken polyclonal anti-peptide antibody; 1:4,000), MBP (Millipore Sigma; catalog # AB980; anti-myelin basic protein antibody; 1:250), or MAG (Millipore Sigma; catalog # PA5–30087; polyclonal antibody; 1:500) antibodies and visualized with Amersham ECL Reagant (GE Healthcare) on ImageQuant LAS 4000 (GE Healthcare). After visualizing for above primary antibodies, blots were stripped with Restore Western Blot Stripping Buffer (Thermo Scientific), reprobed overnight with β-actin (Millipore Sigma; catalog # A5316; monoclonal anti-β-actin antibody; 1:5,000), and again visualized by ECL reagent, as described above.

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Jha M.K., Lee Y., Russell K.A., Yang F., Dastgheyb R.M., Deme P., Ament X.H., Chen W., Liu Y., Guan Y., Polydefkis M.J., Hoke A., Haughey N.J., Rothstein J.D, & Morrison B.M. (2019). Monocarboxylate transporter 1 in Schwann cells contributes to maintenance of sensory nerve myelination during aging. Glia, 68(1), 161-177.

Publication 2019

T-PER Mini-Protean TGX Gels nitrocellulose membranes Amersham ECL Reagant ImageQuant LAS 4000 Restore Western Blot Stripping Buffer β-actin

Actin Anti antibody Antibodies Antibody Aves Buffer Chicken Mct1 Membranes Mice Monoclonal antibody Myelin basic protein Myelin protein zero Nitrocellulose Peptide Perfusion Peripheral nerves Proteins Western blot Western blots

Corresponding Organization : Johns Hopkins University

Other organizations : City University of Hong Kong, Neurological Surgery

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Variable analysis

independent variables

Transcardial perfusion of deeply anesthetized mice with 0.1 M PBS

dependent variables

Protein expression levels of MCT1, P0, MBP, and MAG in peripheral nerves

control variables

Protein loading amount (15–30 μg) for Western blot analysis
Use of β-actin as a loading control

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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