This is a protocol for stripping the probed western membrane to re-probe.
- Probed membrane
- 0.5M Tris-HCl pH6.8
- 1M Tris-HCL pH8.0
- 10% SDS
- Tween 20
- 2.5M NaCl
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.
- 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.
- To make 1L TBST, add 10ml 1M Tris-HCL pH8.0, 60ml 2.5M NaCl, 1ml Tween 20 to water up to 1L.
- 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.
- Rinse the membrane with large volume of DI water for several times.
- Wash the membrane for 5 minutes 3 times in TBST at RT using large volumes of buffer.
- 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.
- Proceed with the standard western blot protocol.
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, 20 July 2011 (EDT)
or instead, discuss this protocol.