b'A Western blot analysis A Western blot analysisStripping Strippingbu\x0eer Initial detection Strip test Reprobed bu\x16er Initial detection Strip test ReprobedRestore RestoreReBlot Plus ReBlot PlusRevitablotRevitablotB Densitometry B DensitometryStripping bu\x0eers: Restore Stripping bu\x16ers: RestoreReBlot Plus ReBlot PlusRevitablot Revitablot120 100110 90100 80Percent of initial signal Percent of initial signal9080 7070 6060 5050 4040 3030202010 0 100 Initial Stripped Reprobed 0 Initial Stripped ReprobedFigure 49. Western blot stripping and reprobing performance ofFigure 50. Western blot stripping and reprobing performance of three three stripping buffers on nitrocellulose. HeLa cell lysate was dilutedstripping buffers on PVDF. HeLa cell lysate was diluted to 1 mg/mL in to 1 mg/mL in electrophoresis reducing sample buffer, and 1:1 serialelectrophoresis reducing sample buffer, and 1:1 serial dilutions were made. dilutions were made. Three sets of 10 L per dilution (10 g to 0.31 g ofThree sets of 10 L per dilution (10 g to 0.31 g of total protein) were total protein) were separated by SDS-PAGE and transferred to 0.45 mseparated by SDS-PAGE and transferred to 0.45 m PVDF membrane. nitrocellulose membrane. (A) The membrane was blocked with 5% nonfat(A) The membrane were as blocked with 5% nonfat dry milk in 1X Pierce dry milk in 1X Pierce TBS Tween 20 Buffer and analyzed by western blotTBS Tween 20 Buffer and analyzed by western blot using SuperSignal using SuperSignal West Dura Extended Duration Substrate and imaged.West Dura Extended Duration Substrate and imaged. The membrane was The membrane was probed with Invitrogen Hsp90 Polyclonal Antibody atprobed with Invitrogen Cyclophilin B Polyclonal Antibody at 0.2 g/mL 0.5 g/mL followed by Invitrogen Goat AntiRabbit IgG, HRP Conjugate,followed by Goat AntiRabbit IgG, HRP Conjugate, at 5.7 ng/mL, and at 5.7 ng/mL, and imaged. Following the initial detection, the blot wasimaged. Following the initial detection, the blot was cut into three strips to cut into three strips to separate the serial dilution sets, and each part ofseparate the serial dilution sets, and each part of the blot was stripped, the blot was stripped, according to manufacturers instructions, in eitheraccording to manufacturers instructions, in either Restore Western Blot Restore Western Blot Stripping Buffer (15 minutes at 37C), ReBlot PlusStripping Buffer (15 minutes at 37C), ReBlot Plus Stripping Solution Stripping Solution (MilliporeSigma; 15 minutes at room temperature), or(MilliporeSigma; 15 minutes at room temperature), or Revitablot Western Revitablot Western Blot Stripping Buffer (Rockland ImmunochemicalsBlot Stripping Buffer (Rockland Immunochemicals Inc.; 15 minutes at Inc.; 15 minutes at room temperature). After the stripping procedure, theroom temperature). After the stripping procedure, the membrane strips membrane strips were washed in 1X PBS Tween 20 buffer and incubatedwere washed in 1X PBS Tween 20 buffer and incubated with the substrate with the substrate and imaged. The membrane strips were reblocked,and imaged. The membrane strips were reblocked, and the western blot and the western blot procedure was repeated as described above.procedure was repeated as described above. (B) Densitometry analysis (B) Densitometry analysis shows that Restore Western Blot Strippingshows that Restore Western Blot Stripping Buffer permitted complete Buffer permitted complete signal removal and maintained nearly identicalsignal removal and maintained nearly identical levels of detection between levels of detection between the initial and reprobed western blot analysis. the initial and reprobed western blot analysis.72'