Stripping and reprobing blots for similar molecular weight targets with Restore Western Blot Stripping Buffer
Western blot stripping and reprobing performance of three stripping buffers on PVDF
HeLa cell lysate was diluted to 1mg/mL in electrophoresis reducing sample buffer and 1:1 serial dilutions were made. Three sets of 10µL/dilution (10µg to 0.31µg of total protein) were separated by SDS-PAGE and the protein transferred to 0.45µm nitrocellulose membrane (Part No. 88018). A. The membrane was blocked with 5% non-fat dry milk in 1X TBS Tween-20 Buffer (Part No. 28360) and analyzed by Western blot using SuperSignal West Dura Extended Duration Substrate (Part No. 34076) and the myECL Imager (3x3 binning) (Part No. 62236). The membrane was probed with Anti-Hsp90 Polyclonal Antibody (Part No. PA3-013) at 0.5µg/mL followed by Goat anti-Rabbit Horseradish Peroxidase Conjugate (Part No. 31460) at 5.7ng/mL and imaged. Following the initial detection, the blot was cut into three strips to separate the serial dilution sets and each part of the blot was stripped, according to manufacturer’s instructions, in either Restore Western Blot Stripping Buffer (Part No. 21059) (15 minutes at 37°C), Reblot Plus Stripping Solution (EMD Millipore, Part No. 2502) (15 minutes at room temperature) or Revitablot™ Western Blot Stripping Buffer (Rockland Immunochemicals Inc., Part No. MB-085-0050) (15 minutes at room temperature)s. After the stripping procedure, the membrane strips were washed in 1X PBS Tween-20 buffer and incubated with the substrate and imaged. The membrane strips were reblocked and the Western blot procedure repeated as described above. B. Densitometry analysis shows that the Restore Stripping Buffer permitted both complete signal removal and maintained nearly identical levels of detection between the initial and reprobed Western blot analysis.