Western Blot Stripping Buffer:

Making your own western blot stripping buffer is easy and affordable. We essentially always use PVDF membranes for our western blots because of their durability and low background for a variety of primary antibodies. PVDF membranes are useful because they can be stripped and re-probed many times to analyze multiple protein targets on the same membrane. This is a time and money saver because it allows us to look at multiple targets, without having to run multiple gels on the same samples. It is also a more technically sound practice to look at various proteins within one blot, rather than comparing between multiple blots (I’m talking about a single experiment. Of course, you’ll want to run new gels for biological replicates).

The recipe for the western blot stripping buffer we use is:

  • 60 mM Tris-HCl pH 6.8
  • .7% β-mercaptoethanol
  • 2% SDS

To make 500 ml of western blot stripping buffer:

  • 30 ml of Tris-HCl pH 6.8
  • 3.5 ml of β-mercaptoethanol
  • 50 ml of 20% SDS
  • Add 416.5 ml of ultrapure water.

 Western Blot Stripping Protocol:

  • After developing the previous blot, remove the membrane from the cassette and place it in TBST to remove ECL.
  • Pour a small amount of western blot stripping buffer into a closed container (under hood because of β-mercaptoethanol). Usually, 5-10 ml is sufficient depending on the size of the container.
  • Place the western blot membrane into the stripping buffer and incubate in a water bath at 50°C for 30 minutes under gentle agitation.
  • Remove the PVDF membrane from the western stripping solution and place it into TBST. Be sure to dispose of the stripping solution properly because it contains  β-mercaptoethanol.
  • Wash the membrane 5 times in TBST under gentle agitation to remove all traces of the western blot stripping buffer.
  • Proceed to the blocking step of the western blot protocol.
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