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91925
Western Blot Stripping Buffer (5X)
WB & IP Reagents
Buffer

Western Blot Stripping Buffer (5X) #91925

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Western Blot Stripping Buffer (5X): Image 1
Western blot analysis of extracts from serum-starved Jurkat cells, treated with LY294002 #9901 or Calyculin A #9902, using Akt (pan) (C67E7) Rabbit mAb #4691 before (left) and after (right) incubating the blot in Western Blot Stripping Buffer (1X) and then reprobing with secondary antibodies to show complete stripping of the membranes.
Western Blot Stripping Buffer (5X): Image 2
Western blot analysis of extracts from serum-starved Jurkat cells, treated with LY294002 #9901 or Calyculin A #9902, using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb #4060 before (left) and after (right) incubating the blot in Western Blot Stripping Buffer (1X) and then reprobing with secondary antibodies to show complete stripping of the membranes.

Storage

Store Western Blot Stripping Buffer (5X) at room temperature. This product is stable for at least 12 months.

Protocol

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Stripping Buffer Protocol

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

SUPPLIED REAGENTS:

  1. Stripping Buffer (#91925): To prepare 20mL of 1X Stripping Buffer, combine 4mL 5X Stripping buffer with 140uL 2-mercaptoethanol (0.1M final concentration) and 15.86mL RODI water.

ADDITIONAL REAGENTS (NOT SUPPLIED):

  1. 10X Tris Buffered Saline with Tween® 20 (TBST) (#9997): To prepare 1 L 1X TBST: add 100 ml 10X TBST to 900 ml dH2O, mix.
  2. 2-mercaptoethanol

B. Stripping Membranes

NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.

NOTE: This protocol begins after membrane exposure to film or digital imager. Best results are obtained if the membrane is not allowed to dry. Membranes can be stored in 1X TBST at 4℃.

NOTE: Quantitative comparison of targets before and after stripping is not recommended due to the removal of small amounts of membrane-bound protein with each round of stripping.

  1. Wash three times for 5 min each with 15 ml of 1X TBST at room temperature.
  2. Incubate membrane in 20 ml of Stripping Buffer for 45 minutes to 1 hr at 50℃ with slight agitation, if available. Depending on antibody signal strength, optimization of incubation time and temperature may be required. The incubation time can be adjusted up to 2 hours while the incubation temperature can be adjusted to 70℃ if needed.
  3. Wash five times for 5 min each with 15 ml of TBST at room temperature.
  4. (Optional) To check the efficiency of the stripping and confirm that the original signal has been removed, incubate the membrane with the appropriate secondary antibody and proceed with protein detection using recommended detection reagents. If stripping is complete, continue by washing the membrane five times for 5 min each with 15 ml of TBST at room temperature. If stripping is insufficient, repeat steps 2 through 4.
  5. The membrane is ready for reuse. Proceed with the blocking step before adding the primary antibody.

Protocol Id: 2564

Product Description

Western Blot Stripping Buffer (5X) allows for quick and effective removal of both primary and secondary antibodies from nitrocellulose membranes. This can be done without damaging the immobilized antigen, enabling the membranes to be reprobed with alternative antibodies or concentrations for further western blot analysis. This product should be diluted to 1X using RODI-water, and 2-mercaptoethanol should be added right before use. Completely submerge the membrane in Western Blot Stripping Buffer (1X). Optimization of incubation time or temperature may be required to achieve desired results.

Background

Having to re-run gels and repeat western blotting efforts is costly and time-consuming. Western Blot Stripping Buffer (5X) enables the membranes to be reused with the added benefit of not having to re-load expensive or limited samples into the gel. Allowing for the detection of a different target with an alternative primary antibody or by reprobing with a different concentration of a primary antibody saves valuable time.

Limited Uses

Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
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U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.
To Purchase # 91925
Cat. # Size Qty. Price
91925S
100 ml  (25 western blots) $ 109