Membrane Stripping Reprobing

Membrane Stripping Reprobing Protocol and information for western blots.

Membrane Stripping and Reprobing

It is easier and more convenient to strip and reporbe a blot with more than one antibody than to run another SDS-PAGE gel with the samples

In addition, loading controls and total protein data is often needed especially when you are analyzing phosphorylation of a phosphor-protein.

These must all be completed on the same membrane to eliminate any factors

Stripping and reprobing allow one to maximize data while minimizing time spent.

The definition of stripping and reprobing a blot is removing or dissociating the antibodies off of the membrane blot so that the blot can be reprobed with a new set of detection antibodies

Limitations of Membrane Stripping for Western Blots

Stripping and reprobing have their Limitations

Harsh stripping not only strips antibodies off of the membrane but usually some of your protein of interest as well.

Sometimes even all the signal is lost with stripping. This is especially the case with phosphor-proteins

Always probe for phosphorylation FIRST and then stripand reprobe for total protein. This is due to the fact that phosphor-proteins always have a weaker signal and are more liable to damage. Total protein signal is usually much higher and stronger allowing you to detect it later after stripping.

As much as ¼ of your protein of interest may be lost or damaged after stripping. Sometimes signal of your target protein will be completely gone.

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Related Items:

• History Western Blot
• Western Blot
• Western Blot Blocking
• Western Blot Detection Methods
• Western Blot Protein Sample Preparation Protocol