Griffin:Membrane Stripping and Reprobing

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Following detection of proteins with enhanced chemiluminescence (ECL), membranes can be stripped of bound antibody and reprobed with a different antibody. If you plan on stripping and reprobing the membrane, the membrane should be stored moist and wrapped in plastic wrap at 2-8°C after each immunodetection.

I) Submerge the membrane in stripping buffer:

62.5 mM Tris-HCl, pH 6.7, 100 mM beta-mercaptoethanol, 2% SDS. Incubate at 50°C for 30 minutes with occasional shaking. If more stringent conditions are needed, this incubate at 70°C.

OR

0.2M glycine pH 2.5 and tween 0.05%. Incubate at 80°C for 20 minutes with occasional shaking.

  • The key to a successful stripping and reprobing is to rinse the membrane until there is no beta-mercaptoethanol odor present. This will require several initial shake rinses into a proper waste storage container, followed by rinsing under milli-q water for a few minutes at a sink, then a few more rinses in TBS or PBS. Check for the odor and when it is gone, the membrane is ready.

II) Wash the membrane twice for 10 minutes each, at room temperature, in 1x TBS, 0.05% Tween-20. Use a large volume (10-20 ml) of buffer for each wash.

III) Block the membrane for 1 hour at room temperature, or overnight at 4°C, in 1x TBS, 5% milk, 0.05% Tween-20.

Comments:

The western blotting procedures are the same for PVDF or nitrocellulose, however the handling of these membranes are different prior to- and during- transfer of proteins from the SDS-PAGE gel to the membrane.

Nitrocellulose exhibits the highest sensitivity with very low backgrounds in all transfers, especially in protein blotting. Unlike PVDF, nitrocellulose wets out naturally, does not require methanol, and will not turn hydrophobic during the transfer process. Nitrocellulose is very easily blocked and does not need the many blocking steps required with PVDF.

Protocols for Western Blotting with PVDF and Nitrocellulose are the same with a few exceptions. PVDF is hydrophobic and therefore should be prewet in methanol before it is used. Wet the membrane in methanol for 15 seconds. Membrane should uniformly change from opaque to semi-transparent. Carefully place the membrane in ultrapure water and soak for 2 minutes. Then carefully place the membrane in transfer buffer and let equilibrate for at least 5 minutes.

Another change to note is that the SDS tolerances are not equivalent for PVDF and Nitrocellulose. The binding of protein to PVDF is much more sensitive to SDS levels. Too much SDS can inhibit the protein's ability to bind to the PVDF and can, in fact, help proteins already bound to the membrane to slip off. SDS levels should never exceed 0.05% for PVDF. and and

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