IMPORTANT: Optimization of incubation time is essential for bets results.
IMPORTANT : If the blot cannot be stripped immediately after chemiluminescence detection, the blot can be stored in PBS or TBST at 4oC until the stripping procedure is to be preformed.
1. Place the blot to be stripped in the Stripping Buffer. Add Stripping Buffer, to fully immerse the blot.
2. Incubate the blot in the Striping buffer for 5-15 min at room temperature with strong shaking.
IMPORTANT: In general, higher affinity antibodies or large quantities of detected protein will require longer incubation time for stripping.
3. Empty the Stripping Buffer.
4. To wash, add 0.3 ml of dH2O and shake vigorously.
5. Repeat Steps 4 five more times.
Complete removal of the HRP label monitoring: After Step 5 incubate the membrane with fresh chemiluminescence reagents and expose to film. If no signal is detected with a 5 min exposure, the HRP conjugate has been successfully removed from the antigen or primary antibody
Complete removal of primary label antibody monitoring: After Step 5 incubate the membrane with the HRP-labeled secondary antibody, followed by a wash in wash buffer. Incubate the membrane with fresh chemiluminescence reagents and expose to film. If no signal is detected with a 5 min exposure, the primary antibody has been successfully removed from the antigen.
IMPORTANT: Analysis of the successful removing of immunoprobes is recommended to prevent removal of the antigen or the unsuccessful removal of the antibodies.
6. If signal is detected in the two experiments describe above, place the blot back into Stripping Buffer for additional 5-15 min.
7. After it has been determined that the membrane is free of the immunodetection reagents, a second immunoprobing can take place. Start the second immunoprobing with reblocking of the blot.
IMPORTANT: The blot can be stripped up to 5 times. However, longer exposure times or more sensitive chemiluminescence substrate. Actually, reprobings may result in a decrease in signal if antigen is labile. Analysis of the individual system is required.