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. Repeated processing of the same membrane with stripping buffer will result in the weakening of the protein signal. It is not advisable to reuse the membrane too many times. Generally, one membrane can be used 2 to 3 times.

WSHTBio’s Western Stripping Buffer (alkaline) mainly release the bound of primary and secondary antibodies by alkalinity without the loss of proteins on the membrane. It is suitable for the reuse of blot membranes after western chemiluminescence detection (such as ECL), especially suitable for PVDF membranes, followed by nitrocellulose membranes. It only takes about 15-20 minutes to achieve the reuse of blot membrane, and then subsequent operations such as blocking can be performed. This product is not suitable for western detection with non-chemiluminescent substrates (such as DAB, NBT/BCIP).

Storage: Store at room temperature away from light, valid for 12 months.

Protocol (for reference only):

1. After chemiluminescent detection, wash blot membrane for 5 minutes in distilled water to remove the chemiluminescent substrate.
2. Decant the distilled water and add sufficient western stripping buffer to cover the blot membrane.
3. Incubate for 5 minutes with gentle shaking.
4. Decant stripping buffer and remove the buffer as much as possible, dry the blot membrane with filter paper if necessary.
5. Wash the blot membrane 2-3 times for 3-5 minutes each in distilled water with gentle shaking.
6. Add blocking buffer (casein or skim milk is recommended as blocking buffer) and proceed to substrate steps of western blotting.

Notes:

1. It is recommended to use 5% skim milk as blocking buffer for horseradish peroxidase-conjugated antibodies, and use casein as blocking buffer for alkaline phosphatase-labeled antibodies.
2. Western stripping buffer (alkaline) is especially suitable for the regeneration of PVDF membranes, the regeneration effect of nitrocellulose membranes is not as good as that of PVDF membranes.
3. Western stripping buffer (alkaline) 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).
4. Western stripping buffer (alkaline) is slightly corrosive, please pay attention to protection when operating.
5. For your safety and health, please wear a lab coat and disposable gloves.
6. For research use only. Not for use in diagnostic procedures.