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Stripping and Reprobing Protocol

Stripping refers to the method of removing a primary and secondary antibody from a western blot (WB) 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. A blot may be stripped and reprobed several times to visualize other proteins or to optimize detection of a protein without the need for multiple gels and transfers. Two methods are normally used to strip membrane, using heat and detergent to release antibodies or using low pH to inactivate the antigen binding site of the antibody.

Advantages of Striping and Reprobing Blots

Save Sample If protein mixture is rare or valuable, reprobing conserves the sample and allows the membrane to be analyzed with different antibodies.
Save Time It is time-consuming to run another SDS-PAGE and then transfer the proteins to another membrane. Striping and reprobing blots save much time.
Saves Money By reusing the same blot, you save money on the costs of the protein sample, gel, membrane.
Easier Assay Optimization Optimization is achieved easily by stripping the membrane and reprobing with a different antibody concentration.
Quickly Confirm Atypical Results When immunoblot results are not as expected, reprobing allows the use of the same protein sample without going back to gel electrophoresis.
Correct Mistakes Immunoblotting requires many steps, providing ample opportunity for mistakes to occur. By stripping the membrane, the blot can be reused.

The key to stripping a membrane is to use conditions that cause the release of antibody from the antigen without causing a significant amount of antigen to be released from the membrane. Various protocols have been developed to accomplish this purpose. During the stripping procedure, some amount of antigen is inevitably lost from the membrane. It is important to minimize this loss by stripping the antibody under gentle conditions. There is no guaranteed method to remove every antibody while preserving the antigen.

Stripping and Reprobing Protocol

Mild Stripping (Stripping by Low pH)

  1. Mild stripping buffer recipe
    • 25 mM glycine-HCl
    • 1% SDS
    • Adjust pH to 2

  2. Protocol
    • Rinse membrane in water to remove excess chemiluminescent substrate on the membrane.
    • Incubate the membrane protein-side up in the stripping buffer with agitation, for 10-20 minutes at RT.
    • Note: Ensure the volume of the stripping buffer is enough to fully cover the membrane. Optimization of both incubation time and temperature is needed.

    • Wash the membrane 3 times with agitation for 5 minutes each in TBS-T.
    • Proceed to the blocking step of the immunoblotting protocol to reprobe the blot with a second antibody.

Stringent Stripping (Stripping by Heat and Detergent)

  1. Stringent stripping buffer recipe
    • 100 mM 2-mercaptoethanol
    • 2% SDS
    • 62.5 mM Tris-HCl
    • Adjust pH to 6.7

  2. Protocol
    • Rinse membrane in water to remove excess chemiluminescent substrate on the membrane.
    • Incubate the membrane protein-side up in the stripping buffer with gentle agitation, for 30 minutes at 50 °C in a fume hood.
    • Note: Ensure the volume of the stripping buffer is enough to fully cover the membrane.

    • Wash the membrane 5 times with agitation for 5 minutes each in TBS-T.
    • Proceed to the blocking step of the immunoblotting protocol to reprobe the blot with a second antibody.

More WB Resource

Western Blot Protocols & Troubleshooting & Guide

Western Blot Protocol

Western Blot Troubleshooting

Sample Preparation in Western Blot Assay

Protein Transfer from Gel to Membrane in Western Blot Assay

Running an SDS-PAGE Gel in Western Blot Assay

Western Blot Illustrated Assay

Western Blot Storage Protocol

For research use only. Not intended for any clinical use.