Introduction:

Western stripping buffer can remove primary and secondary antibodies from membranes, enable the reuse of membranes so that they can be reprobed with different primary antibodies and detected with chemiluminescent substrates again, which can effectively simplify the western blot optimization process.

WSHTBio’s Western Stripping Buffer (neutral) adopts a neutral washing formula for removing bound primary and secondary antibodies from membranes without removing or damaging the immobilized antigen. It eliminates time and cost waste by allowing the reuse of nitrocellulose or PVDF blots to detect different targets with different primary antibodies without the need to re-runs gels and transfer.

Storage: Store at 2~8°C, or -20°C if not in use for a long time.

Protocol (for reference only):

1. After chemiluminescent detection, take out the exposed blot membrane and add sufficient western stripping buffer to completely cover the membrane (about 15 mL for 8.5 cm×5.5 cm membrane).
2. Incubate at room temperature for about 15 minutes.

Note: Optimization of both incubation time and temperature is essential for best results. For some antibodies, room temperature incubation is sufficient. However, high-affinity antibodies or saturated blots (excess secondary antibody) may require incubation for an additional 5 to 10 minutes at 37°C. 

3. Decant the stripping buffer and wash membrane 3 times for 5 minutes each in 15 mL washing buffer (e.g., TBS) with shaking at room temperature. 
4. In order to detect whether the stripping of the enzyme-labeled secondary antibody is complete, incubate the membrane with new chemiluminescent substrate. If no signal is detected using a 5-minute exposure, the secondary antibody has been successfully removed from the antigen or primary antibody
5. If signal is detected in step 4, incubate blot in Stripping Buffer for an additional 5 to 15 minutes and repeat step 2 and step3.

Note: 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.
7. Block the membrane in blocking buffer before continuing with the second immunoprobing experiment.

Notes:

1. When performing multiple strippings and reprobings for different antigens, it is recommended to probe for low-abundant proteins first.
2. Western stripping buffer (neutral) is suitable for primary and secondary antibody elution from membranes that were detected by ECL and other similar chemiluminescent methods. It cannot be used for membranes after western detection with non-chemiluminescent substrates (such as DAB, NBT/BCIP).
3. For your safety and health, please wear a lab coat and disposable gloves.
4. For research use only. Not for use in diagnostic procedures.