McClean:Membrane Stripping Mild
This is a protocol for stripping the probed western membrane to re-probe. This was successfully used by MNM to sequentially probe for Hog1-PP, beta-actin, and total Hog1 for Dexter, et al 2014.
- 15g glycine
- 1g SDS
- 10ml Tween20
- PBS (Phosphate-buffered saline)
- TBST (Tris-buffered saline and Tween20)
- Combine glycine, SDS, 10ml Tween20 and 800ml ultrapure water
- pH the solution to 2.2 with concentrated HCl
- Add ultrapure water to bring solution up to 1L
- Submerge the membrane in stripping buffer. Incubate at room temperature with rotation for 10 minutes.
- Remove the stripping buffer and repeat step 1.
- Remove the stripping buffer and incubate with PBS at room temperature with rotation for 10 minutes.
- Remove the PBS and repeat step 3.
- Remove the PBS and incubate with TBST with rotation for 5 minutes.
- Remove the TBST and wash with TBST with rotation for 5 minutes.
- Continue on to (re-) blocking the membrane.
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
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- Megan N McClean 14:01, 18 November 2014 (EDT)
or instead, discuss this protocol.