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Which Western Blot Membranes Can Be Stripped? How Many Times Can You Strip A Membrane And Reprobe? Western Blot Stripping Protocol and stripping buffer recipe. Information on Western Blot Stripping and how to strip westernblots.
Which Western Blot Membranes Can Be Stripped?
A major difference between nylon, nitrocellulose and PVDF membranes is the ability to support the removal or "stripping" of antibodies off of the membrane (obviously with a minimum of general protein stripping!), and thus the subsequent re-use or recylcing of the membrane for subsequent western blot by further antibody probing. There are many methods for stripping nitrocellulose membrane however the sturdier PVDF membranes allow for easier stripping, and you can reuse them (or strip) several times before background noise limits further western blotting. How Many Times Can You Strip A Membrane And Reprobe? Membranes can be stripped and reprobed as long as you can get a signal, however it seems membranes may only be stripped up to 3 times before the protein signal becomes too weak. Western Blot Stripping Protocol To strip blots: Shake the blot gently at 50C for 30 min
Wash 2X with TBS-Tween
Re-block in milk for at least 1 hour after stripping
Re-probe blot with primary and secondary antibodies Stripping Buffer Recipe
For 50 mL of stripping buffer:
Add the following:
100 mM 2-mercaptoethanol 0.35 ml
2% SDS 10 ml of 10% SDS
62. 5 mM Tris, pH 6.8 12.5 ml of 0.25 M Tris, pH 6.8
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