Mini transblot electrophoretic transfer cell device

Cells were washed with ice cold PBS and scraped off in lysis buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) containing protease and phosphatase inhibitors (Complete Mini EDTAfree and PhosStop, both Roche Applied Science). Sciatic nerves and whole brains of mice postnatal day 18 (P18) were dissociated in lysis buffer using a TissueRuptor (Qiagen). The lysates were incubated on a rotating wheel at 4°C for 45 min and afterwards cleared from nuclei and debris by centrifugation at 2000x g and 4°C for 5 min.
Separation of proteins were performed by SDS-PAGE using a Mini PROTEAN system (Bio-Rad) or Novex NuPAGE SDS-PAGE Gel system (Life technologies) and transferred onto Roti-PVDF membranes (0.45μm, Roth) using a Mini TransBlot Electrophoretic Transfer Cell device (Bio-Rad). Precision Plus Dual Color Protein Standard (Bio-Rad) was used as a marker. Membranes were blocked with 4% (w/v) milk (Roth) in TBST (50 mM Tris, 150 mM NaCl, pH 7.2, 0.1% (v/v) Tween 20) for 30 min at room temperature. Binding of primary antibodies was carried out either at 4°C over night or for 1h at room temperature. Suitable secondary antibodies (coupled to horseradish peroxidase, Dianova) were incubated for 30min at room temperature. All antibodies were diluted in blocking medium. Bands were analyzed densitometrically using ImageLab Software (Biorad) and MBP Signals were normalized to CNP Signals.

Cells were harvested by scraping in cold lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA pH 7.4, 1% (v/v) Nonidet P-40, 0.25% (w/v) sodium deoxycholate) containing HALT protease and phosphatase inhibitors (Thermo Scientific) and incubating on a rotating wheel at 4°C for 45 min. The lysate was cleared from nuclei and debris by centrifugation at 300×g and 4°C for 10 min.
Proteins were separated by SDS-PAGE using a Mini PROTEAN 3 system (Bio-Rad) and transferred onto PVDF membranes (Immobilion-P, Millipore) using a Mini TransBlot Electrophoretic Transfer Cell device (Bio-Rad). To block unspecific binding, membranes were incubated with 4% (w/v) milk (Roth) in TBST (0.05 M Tris, 0.15 M NaCl, pH 7.2, 0.1% (v/v) Tween20) for 30 min at room temperature. Primary antibody binding was carried out overnight at 4°C and appropriate secondary antibodies (coupled with horseradish peroxidase, Dianova) were incubated for 30 min at room temperature, both in blocking medium.

Cells were washed with ice cold PBS and scraped off in lysis buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) containing protease and phosphatase inhibitors (Complete Mini EDTAfree and PhosStop, both Roche Applied Science). The lysate was incubated on a rotating wheel at 4°C for 45 min and afterward cleared from nuclei and debris by centrifugation at 2000 × g and 4°C for 5 min.
Separation of proteins was performed by SDS-PAGE using a Mini PROTEAN system (Bio-Rad) or Novex NuPAGE SDS-PAGE Gel system (Life Technologies) and transferred onto Roti-PVDF membranes (0.45 μm, Roth) using a Mini TransBlot Electrophoretic Transfer Cell device (Bio-Rad). Precision Plus Protein Standard (Bio-Rad) was used as a marker. Membranes were blocked with 4% (w/v) milk (Roth) in TBST [50 mM Tris, 150 mM NaCl, pH 7.2, 0,1% (v/v) Tween 20] for 30 min at room temperature. Binding of primary antibodies was carried out either at 4°C over night or for 1 h at room temperature. Suitable secondary antibodies (coupled to horseradish peroxidase, Dianova) were incubated for 30 min at room temperature. All antibodies were diluted in blocking medium. Image acquisition was performed in a ChemiDoc XRS system using Imagelab software (Biorad).