Strip time off your research safely and effectively.

Thermo Scientific Restore Western Blot Stripping Buffer safely and effectively removes primary and secondary antibodies from nitrocellulose and PVDF to allow reprobing of chemiluminescent Western blots.

Highlights of Restore Western Blot Stripping Buffer:

• Saves time – no need to re-run gels and blots
• Saves precious sample – re-probe the membrane using the same target sample
• Provides efficient stripping – our formulation is more efficient at stripping antibodies than "homemade" buffers
• Gentle – does not damage the target antigen during stripping allowing efficient re-probing
• Odor-free – no mercaptans means no acrid odors
• Economical – less expensive than other competing stripping buffers

About Thermo Scientific Restore Western Blot Stripping Buffer.

Optimizing assay conditions is the best way to achieve optimum results. However, re-performing gel electrophoresis and immunoblot assays to test new primary antibodies or antibody concentrations is time-consuming and expensive. Thermo Scientific Restore Western Blot Stripping Buffer eliminates this waste when detecting immunoblots with chemiluminescent Western blotting substrates. Restore Western Blot Stripping Buffer allows clean and efficient removal of primary and secondary antibodies from immunoblots without removing the immobilized antigen, allowing blots to be stripped and reprobed with confidence.

Applications for Restore Western Blot Stripping Buffer

• Test different primary antibodies – There is no need to waste precious samples and re-run gels to test different primary antibodies. Simply strip the membrane with Restore Western Blot Stripping Buffer to remove the primary antibody. It takes only 5–15 minutes, depending on the affinity of the primary antibody. After stripping, re-probe with a new primary antibody.
• Optimize assay conditions – Using Thermo Scientific SuperSignal West Substrates, secondary antibody concentrations can be optimized after a single stripping and re-probing cycle. A chemiluminescent detection system is the most sensitive method to detect any reagent still bound after the stripping procedure.

Restore Western Blot Stripping Protocol:

• Wash blot to remove chemiluminescent substrate
• Incubate in Restore Western Blot Stripping Buffer for 5-15 minutes at room temperature
• Remove blot and wash in Wash Buffer
• Block membrane
• Test for sufficient removal of antibodies
• Perform next immunoblot experiment

Strip and reprobe effectively with Thermo Scientific Restore Stripping Buffer. HeLa cell lysate was serially diluted and transferred to membrane. Panel A: The blot was probed for Src. Signal was detected with Thermo Scientific Pierce ECL Chemiluminescent Substrate and a 5 minute film exposure. Panel B: The blot was stripped with Restore Western Blot Stripping Buffer and evaluated by incubating in anti-mouse HRP with subsequent Pierce ECL detection. No signal was detected after a 15 minute film exposure. Panel C: The blot was reprobed for GAPDH and detected with a 1 minute film exposure.

Antibody optimization. Western blots of Interleukin-2 (diluted 20-0.156ng) were detected using SuperSignal West Pico Chemiluminescent Substrate. The first blot (A) used the primary antibody diluted to 1/1,000 (0.5µg/ml) of rat anti-mouse IL-2 and the horseradish peroxidase (HRP)-labeled goat anti-rat secondary antibody (Product # 31470) diluted 1/5,000. The same blot was stripped with Restore Western Blot Stripping Buffer (B) for 5 minutes at room temperature and re-probed (C) with the primary antibody at 1/5,000 and the HRP-secondary conjugate at 1:20,000. Thermo Scientific SuperBlock Blocking Buffer was used for blocking.

References:

1. Kaufmann, S.H., et al. (1987). Anal. Biochem. 161: 89-95.
2. Kaufmann, S.H. and Kellner, U. (1998). Erasure of Western blots after autoradiographic or chemiluminescent detection. In Immunochemical Protocols. Ed. Pound, J.D. Humana Press, Totowa, NJ, 223-235.
3. Schrager, J.A., et al. (2002). J. Biol. Chem. 277: 6137-6142.
4. Sorci, G., et al. (20036). Mol. Cell. Biol. 23: 4870-4881.

Related Products and Links:
Restore PLUS Western Blot Stripping Buffer – stronger formulation for tenacious antibodies
Blocking Buffers – choose from protein-based and protein-free blocking buffers
Chemiluminescent Substrates – compare substrates to the choose the best for your application
CL-XPosure Film – X-ray film for chemiluminescent detection
Background Eliminator for Film – remove excess signal from film to make data clearer