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Stripping and reprobing blots for similar molecular weight targets with Restore Western Blot Stripping Buffer
Stripping and reprobing blots for similar molecular weight targets with Restore Western Blot Stripping Buffer. A431 cell lysate was diluted to 125µg/mL in electrophoresis reducing sample buffer and 1:1 serial dilutions were made. 10µL of each dilution (1250ng to 39ng of total protein) were separated by SDS-PAGE and the protein transferred to 0.45µm nitrocellulose membranes (Part No. 88018). The membrane was blocked with 5% non-fat dry milk in 1X PBS Tween-20 Buffer (Part No. 28352) and analyzed by Western blot using SuperSignal West Dura Extended Duration Substrate (Part No. 34076) and the myECL Imager (3×3 binning) (Part No. 62236). The first target (panel 1) was detected by probing with Anti-PDI Monoclonal Antibody (Part No. MA3-019) at 0.33µg/mL followed by Goat anti-Mouse Horseradish Peroxidase Conjugate (Part No. 31460) at 6.7ng/mL and imaged. Next, the blot was stripped in Restore Western Blot Stripping Buffer (Part No. 21059) for 15 minutes at 37°C, washed in 1X PBS Tween-20, incubated with substrate and imaged to check for stripping efficiency (panel 2). The second target (panel 3) was detected by reblocking the membrane and probing with Anti-Actin Monoclonal Antibody (Part No. MA1-744) at 0.5µg/mL followed by the anti-mouse HRP conjugate at 6.7ng/mL and imaged. The blot was stripped again (panel 4) and then probed for multiple targets (alpha-Tubulin, Part No. 62204 at 0.2µg/mL; PDI as above; and Hsp90, Part No. PA3-013 at 0.14µg/mL), and imaged as described.
