How to optimize stripping buffer for western blotting

  1. Prepare a series of stripping buffers to test for example, Triton X- 100, and SDS at different concentrations.
  2. Strip a set of western blot membranes with each stripping buffer.
  3. Use the same primary and secondary antibodies to re-probe the membranes; optimal stripping buffer will depend on the antibodies being used.
  4. Blots can be reviewed to see which stripping buffer gave the best results.

A few additional suggestions

  • First start by optimizing the incubation time and temperature. Based on the stripping buffer you use the incubation time and temperature will vary. Triton X-100 may only need to be incubated for 15 minutes at room temperature, while SDS may need to be incubated for 30 minutes at 50°C.
  • Once the stripping step is completed, it is important to wash the membrane thoroughly to remove any residual stripping buffer. Thorough washing will help to prevent the buffer from interfering with the re-probing step. Washing steps are best carried out in an automated, reproducible fashion. Either the BlotCycler Mini or Touch are idea for automation of all washing and incubation steps
  • Test different stripping buffers. The best way to optimize stripping buffer is to test different buffers and see which one works best for your specific antibodies.
  • Use a fresh stripping buffer for each blot. This will help to ensure that the buffer is not contaminated with antibodies or other proteins.
  • Warm the stripping buffer before using it. This will help to speed up the stripping process.
  • Use gentle agitation when incubating the membrane in the stripping buffer. This will help with even distribution of buffer across the membrane. Ideal distribution of buffer can be achieved using the BlotCycler. We recommend our optimized stripping buffer, https://precisionbiosystems.com/shop/precision-stripping-buffer/

 

Need more information: https://www.blotcycler.com/