Western Membrane Stripping

The complete removal of primary and secondary antibodies from the membrane is possible following the protocol outlined below. The membranes may be stripped of bound antibodies and re-probed several times. Membranes should be stored wet wrapped in SaranWrap in a refrigerator (2–8 °C) after each immuno-detection.

1. Submerge the membrane in stripping buffer (100 mM 2-Mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl pH 6.8) and incubate at 50°C for 30 minutes with occasional agitation. If more stringent conditions are required the incubation can be performed at 70°C or the incubation time increased.

2. Wash the membrane for 10 minutes in PBS-T at RT using large volumes of buffer.

3. Probe the membrane with the secondary antibody to verify the removal of the previous signal.

4. Block the membrane in 5% non-fat dried milk in PBS-T for 1 hour at RT and proceed with the standard western blot protocol.

Reagents:

Stripping Buffer (25mL) 100mM b-Mercaptoethanol (175 uL of liquid b-ME – 14.3M) 2%SDS (5 mL of 10%SDS) 62.5 mM Tris-HCl pH 6.8 (3.125 mL of 4x stacking gel solution)

PBS-T (1L) 1xPBS (100 mL of 10xPBS) 0.1% Tween-20 (1mL Tween-20)