- Data Analysis
- Other Protocols
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.