INSTRUCTIONS

Restore Western Blot Stripping Buffer

21059
Number
21059 21062

3747 N. Meridian Road P.O. Box 117 Rockford, IL 61105

21062
Description
Restore Western Blot Stripping Buffer, 500 ml Restore Western Blot Stripping Buffer, 30 ml

0887.1

Storage: Upon receipt store at room temperature or 4C. Product shipped at ambient temperature.

Introduction
Nitrocellulose and PVDF membranes that have been probed by Western blotting procedures and detected by chemiluminescent or other nonprecipitating substrates may be stripped and re-probed using Restore Western Blot Stripping Buffer. Western blotting is widely used to detect and compare proteins in complex mixtures, and chemiluminescence has largely replaced colorimetric analysis as the most convenient and sensitive method of detection. One advantage of chemiluminescence is the possibility to strip and reprobe the protein mixture on the membrane. Traditional stripping methods use conditions that are effective for only low-affinity antibody-antigen interactions or are so harsh conditions that they tend to adversely alter the antigen for subsequent immunoprobing.1,2 Restore Western Blot Stripping Buffer provides a generally robust but gentle method for stripping primary and secondary antibodies from blots to enable several reprobings on the same membrane. Restore Western Blot Stripping Buffer is ideal for use with SuperSignal West Chemiluminescent Substrates. Note: Restore Buffer will not dissociate interactions between a biotinylated target protein and avidin conjugate probes.

Additional Materials Required

Western blot, previously blocked, probed and detected with chemiluminescent (i.e., nonprecipitatating) substrate Wash Buffer such as Tris buffered saline (TBS) or phosphate buffered saline (PBS) containing 0.05% Tween-20 Primary and secondary antibodies for both first and second Western blotting experiments Film or CCD camera for detection of chemiluminescent signal

Procedure for Stripping an Immunoblot

Notes: Blots that cannot be stripped immediately after chemiluminescent detection may be stored in phosphate buffered saline (PBS) at 4C until the stripping procedure can be performed. 1. 2. Warm bottle of Restore Western Blot Stripping Buffer to room temperature. Place the blot to be stripped in Restore Western Blot Stripping Buffer and incubate for 5-15 minutes at room temperature. Use a sufficient volume to ensure that the blot is completely wetted (i.e., approximately 20 ml required for an 8 x 10 cm blot). Note: Optimization of both incubation time and temperature is essential for best results. In general, higher affinity antibodies will require at least 15 minutes of stripping and may require an incubation temperature of 37C. 3. 4. Remove the blot from the Restore Western Blot Stripping Buffer and wash in Wash Buffer. Test for the removal of the immunodetection reagents.

Warranty: Pierce products are warranted to meet stated product specifications and to conform to label descriptions when used and stored properly. Unless otherwise stated, this warranty is limited to one year from date of sale for products used, handled and stored according to Pierce instructions. Pierces sole liability for the product is limited to replacement of the product or refund of the purchase price. Pierce products are supplied for laboratory or manufacturing applications only. They are not intended for medicinal, diagnostic or therapeutic use. Pierce products may not be resold, modified for resale or used to manufacture commercial products without prior written approval from Pierce Biotechnology. Pierce strives for 100% customer satisfaction. If you are not satisfied with the performance of a Pierce product, please contact Pierce or your local distributor.

Test for complete removal of the HRP label (e.g., secondary antibody): Incubate the membrane with new SuperSignal West Working Solution and expose to film. If no signal is detected using a 5-minute exposure, the HRP conjugate has been successfully removed from the antigen or primary antibody. Test for complete removal of the primary antibody: Incubate the membrane with the HRP-labeled secondary antibody, followed by a wash in wash buffer. Incubate in new SuperSignal West Working Solution and expose to film. If no signal is detected with a 5-minute exposure, the primary antibody has been successfully removed from the antigen.

5.

If signal is detected with either test in step 4, return to step 2, stripping for an additional 5-15 minutes. Some antigen/antibody systems require increased temperature and/or longer incubation times to strip them fully. Optimize stripping time and temperature to ensure complete removal of antibodies while preventing damage to the antigen. After determining that the membrane is properly stripped, the second immunoprobing experiment may be performed. Notes: Blot may be stripped and reprobed several times but may require longer exposure times or a more sensitive chemiluminescent substrate. Subsequent reprobings may result in decreased signal if the antigen is labile in Restore Western Blot Stripping Buffer. Analysis of the individual system is required. Reblocking a membrane is usually not necessary after stripping but may be required in some applications.

6.

Related Pierce Products

34080 34075, 34076 34095, 34096 34090, 34091 37520, 37525 37542, 37538 28320 28374 SuperSignal West Pico Chemiluminescent Substrate, 500 ml SuperSignal West Dura Extended Duration Substrate, 100 ml and 200 ml, respectively; includes HRP-Conjugated Goat Anti-Rabbit and HRP-Conjugated Goat Anti-Mouse antibodies SuperSignal West Femto Maximum Sensitivity Substrate, 100 ml and 200 ml, respectively; includes HRP-Conjugated Goat Anti-Rabbit and HRP-Conjugated Goat Anti-Mouse antibodies CL-XPosure Film, clear blue X-ray film, 100 sheets/pkg, 5x7 and 8x10, respectively Blocker BSA (10X), 10% solutions of bovine serum albumin, 200 ml in TBS and 125 ml in PBS, resectively StartingBlock Blocking Buffer, 1 L in TBS and PBS, respectively Surfact-Amps 20, 6 x 10 ml ampules containing pure 10% solutions of Tween-20 Detergent BupH Modified Dulbeccos PBS Packs, 40 packs, each yielding 500 ml of 8 mM sodium phosphate, 2 mM potassium phosphate, 140 mM sodium choride and 10 mM potassium chloride, pH 7.4 when dissolved in 500 ml ultrapure water BupH Tris Buffered Saline Packs, 40 packs, each yielding 500 ml of 25 mM Tris and 150 mM sodium chloride, pH 7.2 when dissolved in 500 ml ultrapure water

28376

References
1. 2. Kaufmann, S.H., Ewing, C.M. and Shaper, J.H. (1987). The erasable Western blot. Anal. Biochem. 161: 89-95. 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., p. 223-35.

Tween is a registered trademark of ICI Americas. The most current versions of all product instructions are available at www.piercenet.com. For a faxed copy, contact customer service (in the USA call 800-874-3723) or your local distributor. Pierce Biotechnology, Inc., 6/2004. Printed in the USA.

In the USA call: 800-8-PIERCE (800-874-3723) or 815-968-0747 Fax: 815-968-7316 or 800-842-5007 www.piercenet.com