Strip time off your research safely and effectively.

Tired of re-running electrophoresis gels and waiting to see your results? Although optimizing assay conditions is the best way to achieve optimum results, re-performing the gel electrophoresis process to test each new primary antibody or antibody concentration is time-consuming and expensive. You can forget about starting over when you use Thermo Scientific Restore Western Blot Stripping Buffer!

Highlights of Restore Western Blot Stripping Buffer:

• Saves time – no need to re-run gels
• Saves precious sample – re-probe the membrane using the same target sample
• Provides efficient removal – proprietary formulation works better than "homemade" buffers
• Gentle formulation – does not damage target protein after stripping and re-probing
• Odor-free – no mercatans means no acrid odors
• Economical – less expensive than other competing stripping buffers

Applications for Restore Western Blot Stripping Buffer

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

Restore PLUS 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 immunoprobing experiment

Re-probing with different antibodies. Western blots of HeLa cell lysate protein (diluted from 750 ng to 83.3 ng) were detected with SuperSignal West Dura Chemiluminescent Substrate. The first blot used polyclonal rabbit anti-JAK-1 primary antibody at 1:2,000 dilution with an HRP-secondary conjugate diluted at 1:350,000. The same blot was stripped for 5 minutes at room temperature in Restore Western Blot Stripping Buffer and then re-probed with purified mouse anti-human Bak monoclonal primary antibody at 1:1,000 with the HRP-secondary conjugate at 1:100,000. Five-percent nonfat milk with 0.05% Tween*-20 was used for blocking.

Reprobing with different antibodies.Antibody optimization study. Western blots of Interleukin-2 (diluted 20-0.156 ng) were detected using SuperSignal West Pico Chemiluminescent Substrate. The first blot (A) used the primary antibody diluted to 1/1,000 (0.5 µg/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
Restore Fluorescent Western Blot Stripping Buffer – formulated for fluorescent blotting on PVDF
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