Samples from two animals were used to validate the proteomics results by one- and two-dimensional western blotting. For this purpose, samples were homogenized and fractionated into membrane, soluble and insoluble proteins, as described above.
Samples for 1D western blot were diluted 1:1 with Laemmli buffer (Bio-Rad, Hercules, CA) and 40 μg of protein were loaded on 10% SDS-PAGE gels. Samples for 2D western blots (40 μg of protein) were loaded in 200 μl of rehydration buffer, and proteins were separated by isoelectric point and then by molecular weight, as previously described30 (link).
Proteins from one or two-dimensional gels were transferred onto a low fluorescence PVDF membrane (Millipore, Billerica, MA) in semi-dry conditions at 15 V for 45 minutes, blocked in 5% fat-free powder milk for 2 hours at room temperature, and incubated overnight with mouse monoclonal anti-rat HSP70 antibody (1:1000, Abcam, Cambridge, MA). Membranes were simultaneously incubated with a rabbit polyclonal anti-rat β-Actin (1:3000, Abcam, Cambridge, MA). For detection, either a goat anti-mouse Cy3-labelled secondary antibody (1:3000, GE, Barcelona, Spain) or a goat anti-rabbit Cy5 (1:3000, GE, Barcelona, Spain) were used, and images were acquired using a Molecular Imager FX Pro-plus (BioRad, Hercules, CA) and analysed with QuantityOne or PDQuest software.
Samples for 1D western blot were diluted 1:1 with Laemmli buffer (Bio-Rad, Hercules, CA) and 40 μg of protein were loaded on 10% SDS-PAGE gels. Samples for 2D western blots (40 μg of protein) were loaded in 200 μl of rehydration buffer, and proteins were separated by isoelectric point and then by molecular weight, as previously described30 (link).
Proteins from one or two-dimensional gels were transferred onto a low fluorescence PVDF membrane (Millipore, Billerica, MA) in semi-dry conditions at 15 V for 45 minutes, blocked in 5% fat-free powder milk for 2 hours at room temperature, and incubated overnight with mouse monoclonal anti-rat HSP70 antibody (1:1000, Abcam, Cambridge, MA). Membranes were simultaneously incubated with a rabbit polyclonal anti-rat β-Actin (1:3000, Abcam, Cambridge, MA). For detection, either a goat anti-mouse Cy3-labelled secondary antibody (1:3000, GE, Barcelona, Spain) or a goat anti-rabbit Cy5 (1:3000, GE, Barcelona, Spain) were used, and images were acquired using a Molecular Imager FX Pro-plus (BioRad, Hercules, CA) and analysed with QuantityOne or PDQuest software.