McClean:Membrane stripping

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Contents

Overview

This is a protocol for stripping the probed western membrane to re-probe.

Materials

  • Probed membrane
  • b-Mercaptoethanol
  • 0.5M Tris-HCl pH6.8
  • 1M Tris-HCL pH8.0
  • 10% SDS
  • Tween 20
  • 2.5M NaCl


Stock Solutions

0.5M Tris-HCl pH6.8

  • Add 60.5g Tris base to 700ml milliQ water, adjust the pH to 6.8 with HCl and top with water to 1L.

1M Tris-HCl pH8.0

  • Add 121g Tris base to 700ml milliQ water, adjust the pH to 8.0 with HCl and top with water to 1L.

Stripping buffer

  • To make 100ml stripping buffer, add 20ml 10% SDS, 12.5ml 0.5M Tris-HCl pH6.8, 67.5ml milliQ water and 800ul b-Mercaptoethanol.

TBST

  • To make 1L TBST, add 10ml 1M Tris-HCL pH8.0, 60ml 2.5M NaCl, 1ml Tween 20 to water up to 1L.


Protocol

  1. Submerge the membrane in stripping buffer (100 mM 2-Mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl pH 6.8) and incubate at 50°C for 30 minutes with agitation.
  2. Rinse the membrane with large volume of DI water for several times.
  3. Wash the membrane for 5 minutes 3 times in TBST at RT using large volumes of buffer.
  4. Block the membrane in 5% non-fat dried milk in TBST for 1 hour and wash the membrane for 5 minutes 4 times in TBST at RT.
  5. Proceed with the standard western blot protocol.




Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

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References

Gietz, R.D. and R.A. Woods. (2002) TRANSFORMATION OF YEAST BY THE Liac/SS CARRIER DNA/PEG METHOD. Methods in Enzymology 350: 87-96.

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