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Restore Western Blot Stripping Buffer


Strip time off your research safely and effectively.

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. Restore Stripping Buffer provides clean and efficient removal of primary and secondary antibodies from immunoblots without removing or damaging the immobilized antigen allowing 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 with reagents such as Thermo Scientific SuperSignal Substrates for horse radish peroxidase is the most common and sensitive method in use today. Because these substrates do not precipitate and bind to membrane surfaces permanently, Western blots detected by chemiluminescence can be stripped with reagents that remove affinity-bound primary and secondary antibodies. To be effective, a stripping buffer must be strong enough to disassociate bound antibodies but gentle enough to leave the transferred target proteins intact on the nitrocellulose or PVDF membrane. Restore Western Blot Stripping Buffer has these characteristics.

By stripping and reprobing, there is no need to waste rare or costly samples by running multiple gels in order to probe for different targets. A single membrane from one gel can be stripped with Restore Western Blot Stripping Buffer to remove the primary antibodies. Stripping the blot takes only 15 to 30 minutes, depending on the affinity of the primary antibody. After stripping, block and reprobe with a new primary antibody. Alternatively, a blot can be stripped and reprobed with adjusted antibody concentrations to optimize conditions after obtaining initialy poor results.

Optimize antibody dilutions using Restore Western Blot Stripping Buffer. Hep G2 (hepatocellular carcinoma) cell lysate was diluted to 1mg/mL in electrophoresis reducing sample buffer followed by 1:1 serial dilutions. 20µL of each concentration (20µg to 0.156µg of total protein) were separated by SDS-PAGE and then protein transferred to 0.45µm nitrocellulose membrane (Part No. 88018). 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). Following the blocking step, the membrane was initially probed with Anti-PDI Monoclonal Antibody (Part No. MA3-019) at 1µg/mL followed by Goat anti-Mouse Horseradish Peroxidase Conjugate (Part No. 32430) at 6.67ng/mL and then detected with substrate and exposure for 1 minute to CL-XPosure Film (Part No. 34091) (panel 1). The blot was then stripped (15 minutes at 37°C) in Restore Western Blot Stripping Buffer (Part No. 21059), washed in 1X PBS Tween-20 buffer and incubated with the substrate to check for stripping efficiency after 5 minutes to film (panel 2). The blot was re-blocked and reprobed with the primary antibody at 0.5µg/mL and secondary antibody at 4ng/mL and detected (panel 3). The blot was stripped again (panel 4) and the membrane reprobed (panel 5) with primary antibody at 0.1µg/mL and secondary antibody at 6.67ng/mL.

A. Western Blot Analysis
B. Densitometry
Western blot stripping and reprobing performance of three stripping buffers on nitrocellulose. 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.

A. Western Blot Analysis
B. Densitometry
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 PVDF membrane (Part No. 88518). A. The membrane were as 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-Cyclophilin B Polyclonal Antibody (Part No. PA1-027A) at 0.2µg/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 east part of the blot was stripped 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) according to manufacturer’s instructions. 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.

Stripping and reprobing blots for similar molecular weight targets with Restore Western Blot Stripping Buffer. A431 cell lysate was diluted to 125µg/mL in electrophoresis reducing sample buffer and 1:1 serial dilutions were made. 10µL of each dilution (1250ng to 39ng of total protein) were separated by SDS-PAGE and the protein transferred to 0.45µm nitrocellulose membranes (Part No. 88018). The membrane was blocked with 5% non-fat dry milk in 1X PBS Tween-20 Buffer (Part No. 28352) and analyzed by Western blot using SuperSignal West Dura Extended Duration Substrate (Part No. 34076) and the myECL Imager (3×3 binning) (Part No. 62236). The first target (panel 1) was detected by probing with Anti-PDI Monoclonal Antibody (Part No. MA3-019) at 0.33µg/mL followed by Goat anti-Mouse Horseradish Peroxidase Conjugate (Part No. 31460) at 6.7ng/mL and imaged. Next, the blot was stripped in Restore Western Blot Stripping Buffer (Part No. 21059) for 15 minutes at 37°C, washed in 1X PBS Tween-20 buffer, incubated with substrate and imaged to check for stripping efficiency (panel 2). The second target (panel 3) was detected by reblocking the membrane and probing with Anti-Actin Monoclonal Antibody (Part No. MA1-744) at 0.5µg/mL followed by the anti-mouse HRP conjugate at 6.7ng/mL and imaged. The blot was stripped again (panel 4) and then probed for multiple targets (alpha-Tubulin, Part No. 62204 at 0.2µg/mL; PDI as above; and Hsp90, Part No. PA3-013 at 0.14µg/mL), and imaged as described.

Protocol Summary:

  • Wash blot to remove chemiluminescent substrate.
  • Incubate blot in Restore Western Blot Stripping Buffer for 5 to 15 minutes at 37°C
    (room temperature is sufficient for some antibodies).
  • Remove blot and wash in Wash Buffer (TBS-T or PBS-T).
  • 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


Ordering Information
 
Product # Description Pkg. Size Instructions MSDS CofA Price
21062 Restore Western Blot Stripping Buffer, Trial Size
Formulation: Proprietary clear, colorless, acidic solution
Sufficient For: 2 mini blots or 160cm2 membrane
30mL Product Instructions for product #21062 Restore Western Blot Stripping Buffer, Trial Size MSDS for product #21062 Restore Western Blot Stripping Buffer, Trial Size Certificate of Analysis for product #21062 Restore Western Blot Stripping Buffer, Trial Size $38.00

Add to cart
21059 Restore Western Blot Stripping Buffer
Formulation: Proprietary clear, colorless, acidic solution
Sufficient For: 25 mini blots or 2000cm2 membrane
500mL Product Instructions for product #21059 Restore Western Blot Stripping Buffer MSDS for product #21059 Restore Western Blot Stripping Buffer Certificate of Analysis for product #21059 Restore Western Blot Stripping Buffer $127.00

Add to cart
21063 Restore Western Blot Stripping Buffer
Formulation: Proprietary clear, colorless, acidic solution
Sufficient For: 250 mini blots or 20,000cm2 membrane
5L Product Instructions for product #21063 Restore Western Blot Stripping Buffer MSDS for product #21063 Restore Western Blot Stripping Buffer Certificate of Analysis for product #21063 Restore Western Blot Stripping Buffer $540.00

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Instructions | MSDS | CofA
Product Instructions | MSDS | CofA  

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