Western blot membrane stripping for reprobing



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Running the SDS-PAGE gel

Transferring protein from gel to membrane

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Membrane stripping and reprobing

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MEMBRANE STRIPPING AND REPROBING

Stripping refers to the method of removing a primary and secondary antibody from a western blot membrane. A single blot can be analyzed sequentially with multiple antibodies by stripping one antibody from the blot and subsequently incubating with an additional antibody. Membrane stripping is often performed as a means to assess the expression of loading control proteins. Following detection of the protein of interest, a membrane can be stripped and reprobed with a loading control antibody to ensure equal expression of the loading control. This practice may also be useful when sample is limited. Two methods to strip your membrane are outlined below. The first method uses heat and detergent to release antibodies and the second uses low pH to inactivate the antigen binding site of the antibody. As a general rule, it is recommended to begin with a milder stripping solution (Stripping with acidic pH, stripping solution #2). If stripping is incomplete and antibody is detected, then a harsher stripping protocol (stripping with heat and detergent) can be used.

Quantitative comparisons of protein expression before and after membrane stripping are not recommended. This is because stripping will remove some sample protein from the membrane.


Buffer Components
Stripping solution #1 100 mM 2-mercaptoethanol
2% SDS
62.5 mM Tris-HCl
Adjust pH to 6.7
Stripping solution #2 25 mM glycine-HCl
1% SDS
Adjust pH to 2


Stripping with heat and detergent protocol

  1. In a fume hood, agitate the blot in stripping solution #1 for 30 minutes at 50˚C

  2. Agitate the blot in 1X PBS for 10 minutes at room temperature. Repeat with fresh buffer.

  3. Proceed to the blocking step of the immunoblotting protocol to reprobe the blot with a second antibody.


Stripping with acidic pH protocol

  1. Agitate the blot in stripping solution #2 for 30 minutes at room temperature.

  2. Agitate the blot in 1X PBS for 10 minutes at room temperature. Repeat with fresh PBS.

  3. Proceed to the blocking step of the immunoblotting protocol to reprobe the blot with a second antibody.



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