Spectra multicolor high range protein ladder

Cell homogenates were collected in RIPA-buffer containing phosphatase and protease inhibitors. Protein extracts were mixed with 5Â sample loading buffer and boiled for 5 min at 95 C. Protein content was determined using the Bradford reagent (Cod. B6916, Sigma-Aldrich Corp.), as previously (Follo et al., 2011) (link). About 30 mg of protein was separated by electrophoresis on a 15% and 6% (depending on the molecular size of the protein of interest) SDS-PAGE gel and then transferred to PVDF membrane (Bio-Rad, Hercules, CA, USA). PageRuler Prestained Protein Ladder (Cod. 26616, Thermo Fisher Scientific Inc. Waltham, MA, USA) and Spectra Multicolor High Range Protein Ladder (cod. 26625, Thermo Fisher Scientific Inc.) were used as Molecular weight markers. The membranes were blocked with 5% non-fat milk in Phosphate Buffer Saline (PBS) with 0.2% Tween-20 for 1 h at room temperature and incubated with specific primary antibodies overnight at 4 C. btubulin was used as homogenate protein loading control. Immunocomplexes were revealed by incubation with an appropriate peroxidase-conjugated secondary antibody (cod. 170515, 1706516, Bio-Rad), followed by peroxidase-induced chemiluminescence reaction (cod. NEL103E001 EA, PerkinElmer, Waltham, MA, USA). Intensity of the bands was estimated by densitometry (Quantity One Software, Bio-Rad or ImageJ software).

One microgram of protein from each fraction was separated on 4%-15% Mini-Protean ® TGX™ precast gel (Bio-Rad Laboratories B.V., Veenendaal, The Netherlands). A Laemmli sample buffer (Bio-Rad Laboratories B.V.) was used for samples analyzed under nonreducing conditions. To obtain reducing conditions, b-mercaptoethanol (Bio-Rad Laboratories B.V.) was added to Laemmli buffer to a final concentration of 355 mM. Prior to loading on the gel, samples were boiled at 95 C for 5 min. The electrophoresis was performed with a Biorad Mini-Protean module and 25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3 (Bio-Rad Laboratories B.V.) as a running buffer. Separation was initiated with electrophoresis at 80 V for 10 min, followed by 120 V for 50 min. Spectra™ Multicolor High Range Protein Ladder (Thermo Fisher Scientific) and Precision Plus Protein™ All Blue Standards (Bio-Rad Laboratories B.V.) were included for molecular weight determination. Silver Stain Plus kit (Bio-Rad Laboratories B.V.) was used for visualization of the protein bands. The images of gels were acquired with a GS-900 densitometer (Bio-Rad Laboratories B.V.) and Image Lab v.5.2.1 software (Bio-Rad Laboratories B.V.).