Membrane stripping-PDF

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.

References

abcam.com/technical