7/29/2019 Stripping Buffer
1/2
INSTRUCTIONS
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Number Description
21059 Restore Western Blot Stripping Buffer, 500 ml
21062 Restore Western Blot Stripping Buffer, 30 ml
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 chemilumi-
nescent 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 chemilumine-
scence 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. Warm bottle of Restore Western Blot Stripping Buffer to room
temperature.
2. Place the blot to be stripped in Restore Western Blot
Stripping Buffer and incubate for 5-15 minutes at roomtemperature.
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 37 C.
3. Remove the blot from the Restore Western Blot Stripping
Buffer and wash in Wash Buffer.
4. Test for the removal of the immunodetection reagents.
3747 N. Meridian Road
P.O. Box 117
Rockford, IL 61105
0887.121059 21062
Restore Western Blot
Stripping Buffer
7/29/2019 Stripping Buffer
2/2
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
2
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
newSuperSignal WestWorking 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.
6. 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.
Related Pierce Products
34080 SuperSignal
West Pico Chemiluminescent Substrate, 500 ml
34075, 34076 SuperSignal
West Dura Extended Duration Substrate, 100 ml and 200 ml,
respectively; includesHRP-Conjugated Goat Anti-Rabbit and
HRP-Conjugated Goat Anti-Mouse antibodies
34095, 34096 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
34090, 34091 CL-XPosure Film, clear blue X-ray film, 100
sheets/pkg, 5x7 and 8x10, respectively
37520, 37525 Blocker BSA (10X), 10% solutions of bovine serum
albumin, 200 ml in TBS and 125 ml in PBS,resectively
37542, 37538 StartingBlock Blocking Buffer, 1 L in TBS and PBS,
respectively
28320 Surfact-Amps
20, 6 x 10 ml ampules containing pure 10% solutions of Tween-20
Detergent
28374 BupH Modified Dulbeccos PBS Packs, 40 packs, each yielding
500 ml of 8 mM sodiumphosphate, 2 mM potassium phosphate, 140 mM
sodium choride and 10 mM potassium chloride, pH7.4 when dissolved
in 500 ml ultrapure water
28376 BupH Tris Buffered Saline Packs, 40 packs, each yielding
500 ml of 25 mM Tris and 150 mMsodium chloride, pH 7.2 when
dissolved in 500 ml ultrapure water
References
1. Kaufmann, S.H., Ewing, C.M. and Shaper, J.H. (1987). The
erasable Western blot. Anal. Biochem.161: 89-95.
2. Kaufmann, S.H. and Kellner, U. (1998). Erasure of Western
blots after autoradiographic or chemiluminescent detection.
InImmunochemical 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.
