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#reprobing

2 public questions tagged with this topic.

Blotting Techniques

What is the role of stripping and reprobing?

Stripping and reprobing constitutes essential procedure to reuse same membrane for detecting multiple different proteins. After initial detection, membrane is incubated in buffers containing SDS, beta-mercaptoethanol or dithiothreitol, and glycine at low pH or elevated temperature to dissociate and remove bound primary and secondary antibody complexes, while target proteins remain immobilized due to strong hydrophobic interactions with PVDF or nitrocellulose. Subsequent reprobing with antibody against different antigen, such as housekeeping protein beta-actin or phosphorylated isoform, allows normalization, conserves limited clinical or fractionated samples, and reduces inter-gel variability.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Blotting Techniques

Which membrane offers highest durability and is suitable for reprobing?

Polyvinylidene difluoride, marketed as PVDF, is a fluoropolymer membrane distinguished by exceptional mechanical toughness, chemical inertness, and thermal stability. Unlike nitrocellulose which becomes brittle upon drying and cannot endure harsh stripping buffers, PVDF remains flexible and intact through exposure to SDS, beta-mercaptoethanol, heat, and low pH glycine treatments. Hydrophobic and dipole interactions confer binding capacity of approximately 170-200 micrograms per square centimeter, much higher than nitrocellulose. Methanol activation is prerequisite. Durability permits sequential probing for multiple antigens, loading controls, and phosphorylated forms from same precious lysate.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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