Mild stripping the PVDF membrane for Odyssey
Aim
PVDF membranes can be stripped to use another antibody detection(re-probing), but after stripped membrane antibody detection efficiency might be reduced.
Materials
PVDF membrane. *DO NOT allow it to dry before, during, or after imaging (keep the blot as wet as possible).
Mild stripping buffer: 25 mM glycine pH 2.0 + 1% SDS
Wash buffer: PBS + 0.1% Tween-20
Methods
1. Incubate blot in stripping buffer for 10-15 minutes at room temperature, with shaking.
2. Replace with fresh stripping buffer and shake an additional 10-15 minutes.
3. Wash in PBS + 0.1% Tween-20 for 5 minutes, with shaking.
4. Rinse with PBS and quickly scan blot at low resolution (337 μm) to see if signal has been removed. *If some signal remains, repeat stripping procedure as needed. Intense bands or strong antibody interactions may require additional incubations in stripping buffer. Frequent changes of stripping buffer are helpful.
5. When ready to begin the next round of detection, re-block for 30-60 minutes and proceed with antibody incubations.
Tips
Avoid overstripping the membrane, as target proteins may be lost from the blot during extended incubations.
Scan membrane to determine when stripping is complete. If overstripping is a problem, try reducing the
amount of SDS in the stripping buffer.
Depending on the strength of the antibody-antigen interactions, you may need to increase the stringency. This
can be achieved by increasing the SDS concentration from 1% to 1.5 - 2%, or by preheating the buffer to
65°C before use. Pour the warmed buffer onto the blot and shake at room temperature or perform stripping
by shaking at 65°C in an oven or water bath. When using elevated temperature, check the blot frequently by
scanning to avoid overstripping.
Date : 2019.03.26
Modified Date :
Author : Reina