Restore Western Blot Stripping Buffer

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

Performing gel electrophoresis and duplicate immunoblot assays to test new primary antibodies or antibody concentrations is time-consuming and expensive. Restore Western Blot Stripping Buffer eliminates this waste when detecting immunoblots with chemiluminescent Western blotting substrates. The reagent allows clean and efficient removal of primary and secondary antibodies from immunoblots without removing or damaging the immobilized antigen. This allows blots to be stripped and reprobed with confidence.

Highlights:

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

Applications:

• Reuse a nitrocellulose or PVDF blot to detect a different target with a different primary antibody
• Reprobe a blot to correct or optimize antibody concentrations that were ineffective the first time

Product Details:

Chemiluminescent Western blot detection, such as with Thermo Scientific SuperSignal Substrates for HRP, is the most common and sensitive method in use today. Because these substrates do not precipitate and permanently bind to membrane surfaces, Western blots detected with by chemiluminescence can be stripped with reagents that remove affinity-bound primary and secondary antibodies.To be effective, a stripping buffer must be be strong enough to disassociate bound antibodies but gentle enough to leave intact the transferred targets on the nitrocellulose or PVDF membrane. Restore Western Blot Stripping Buffer has these characteristics.

There is no need to waste rare or costly samples by running multiple gels to probe for different targets. A single membrane from one gel can be stripped with Restore Western Blot Stripping Buffer to remove the primary antibody. It takes only 5 to 15 minutes, depending on the affinity of the primary antibody. After stripping, re-probe with a new primary antibody. Alternatively, a blot can be stripped and reprobed with adjusted or optimized concentrations of the same antibodies when incorrect amounts were used initially as indicated by poor results.

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 with Thermo Scientific Restore Stripping Buffer. A Western blot of Interleukin-2 (loaded at 20 to 0.156ng per lane) were detected using Thermo Scientific SuperSignal West Pico Chemiluminescent Substrate. The first blot (A) used the primary antibody diluted to 1/1000 (0.5µg/mL) of rat anti-mouse IL-2 and the horseradish peroxidase (HRP)-labeled goat anti-rat secondary antibody (Part No. 31470) diluted 1/5000. 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 1/5000 and the HRP-secondary conjugate at 1:20,000. Thermo Scientific SuperBlock Blocking Buffer was used for blocking.

Protocol Summary:

• Wash blot to remove chemiluminescent substrate
• Incubate blot in Restore Western Blot Stripping Buffer for 5 to 15 minutes at room temperature
  (or incubate at 37°C for high-affinity antibodies)
• Remove blot and wash in Wash Buffer
• Test for sufficient removal of antibodies
• Perform next immunoblot experiment

General References:

1. Baolin Zhang, B, et al. (2003). Mol. Cell. Biol. 23: 5716-5725.
2. Kaufmann, S.H., et al. (1987). Anal. Biochem. 161: 89-95.
3. 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.
4. Lanying Wen, L., et al. (2003). Genetics. 165: 771-779.
5. Schrager, J.A., et al. (2002). J. Biol. Chem. 277: 6137-6142.
6. Skurk, C., et al. (2004). J. Biol. Chem. 277: 6137-6142.
7. Sorci, G., et al. (2003). Mol. Cell. Biol. 23: 4870-4881.

Related Products:

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