Eccles:Western Blot Stripping

=Western Blot Stripping=

Stripping Buffer
4ml 10% SDS (2%) 136.8uL B mercaptoethanol (100mM) 2mL 0.5M Tris pH 6.8 (50mM) 14mL MQH2O


 * Add B mercaptoethanol to buffer last and do it in the fumehood.
 * Heat buffer to 50 degrees C in water bath
 * Incubate membrane in stripping buffer at 50 degrees C for 15min. (I do in 50mL tube in hyb oven in Stephen Robertson's lab - works well). Do not exceed 15min as not only are your previosly bound antibodies stripped from the membrane in this time but also some lysate protein will be stripped.  Exceeding 15min could mean the obvious.
 * Rinse blot multiple times in TBST (do on shaker in fumehood) until the smell of B mercaptoethanol is gone (usually 3 washes is enough)
 * Block and immunostain membrane as usual
 * Probe for your protein of interest first and actin after the strip as actin is prevalent and should be easily detectable after stripping. Lysate protein is lost from the membrane after each strip.