McClean:Membrane Stripping Mild

Overview

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.

Materials

• 15g glycine
• 1g SDS
• 10ml Tween20
• PBS (Phosphate-buffered saline)
• TBST (Tris-buffered saline and Tween20)

Stripping Buffer

• 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

Protocol

1. Submerge the membrane in stripping buffer. Incubate at room temperature with rotation for 10 minutes.
2. Remove the stripping buffer and repeat step 1.
3. Remove the stripping buffer and incubate with PBS at room temperature with rotation for 10 minutes.
4. Remove the PBS and repeat step 3.
5. Remove the PBS and incubate with TBST with rotation for 5 minutes.
6. Remove the TBST and wash with TBST with rotation for 5 minutes.
7. Continue on to (re-) blocking the membrane.

Notes

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References

abcam.com/technical

Contact

• Megan N McClean 14:01, 18 November 2014 (EDT)

or instead, discuss this protocol.