BioProtocols--western4

Procedure for Stripping an Immunoblot using Pierce Restore Stripping Buffer

You also can try our home made stripping buffer--2% Sodium Docecyl Sulfate (SDS), 10mM 2-Mercaptomethanol, 62.5mM Trizma base,pH 6.7--with the same procedure as follows.

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 room temperature. Use a sufficient volume to ensure that the blot is completely wetted (i.e.,approximately 10 ml).

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. Remove the blot from the Restore Western Blot Stripping Buffer and wash in Wash Buffer.

4. Test for complete removal of the HRP label (e.g., secondary antibody): Incubate the membrane with new chemiluminescent detection reagent 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.

5. If signal is detected 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 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.