Western blotting is a commonly used technique for studying protein function and localization. Typically, protein samples are electrophoresed on SDS-PAGE and transferred to a membrane such as nitrocellulose or nylon, where they are probed with specific antibodies. Unlike nucleic acid based technologies, which allow reuse of Southern and Northern blots, it has been difficult to reuse Western blots.
Stripping and reprobing of Western blots offers several advantages:
1) Conservation of samples that are expensive or available only in limited quantities,
2) Analysis of a given blot using several different antibodies, e.g. subtype- or isoform-specific antibodies,
3) Reanalysis of anomalous results and confirmation with the same or a different antibody,
4) Correcting errors in incubation with the wrong antibody,
5) Cost savings in reagents and time by reusing the same blot.
While antigen and antibody based immunoaffinity matrices, such as Sepharose® conjugates, have been reused many times without compromising antigen-antibody reactivity, the need for pH extremes and chaotropic agents has precluded the application of these methods to Western blotting.
The MILLIPORE Re-Blot Plus Western Blot Recycling Kit contains specially formulated solutions that quickly and effectively remove antibodies from Western blots without significantly affecting the immobilized proteins.
Advantages of the Re-Blot Plus Western Blot Recycling Kit include:
- No pungent smelling β-mercaptoethanol is contained in the Antibody Stripping Solution.
- Antibody stripping is done at room temperature. No heating of blots is required.
- Blots can be stripped of antibodies in approximately 15 minutes at room temperature.
- Blots may be reused in 25 minutes.
The MILLIPORE Re-Blot Plus Western Blot Recycling Kit is effective for removal of antibodies from Western blots that have been developed with chemiluminescence or radioactive iodine or other isotopes. It is not recommended for stripping colorimetric substrates (TMB, DAB, 4-chloronapthol, etc.), as it is not possible to effectively remove substrates that precipitate at the reaction site.
The Re-Blot Plus Western Blot Recycling Kit should be used only for qualitative purposes until it has been established by comparative blot analysis that stripping does not quantitatively affect a given antigen.
This product is for research use only; not for diagnostic or in vivo use.
Mild Antibody Stripping Solution (10x) - (1 container, 50 mL).
Strong Antibody Stripping Solution (10x) - (1 container, 50 mL).
Storage and Stability
Kit components should be stored at 4°C upon arrival. Product is stable for 3 to 6 months after receipt. If Antibody Stripping Solution crystallizes upon storage, it may be re-dissolved with gentle warming at 37°C before use.
Note: To prevent reagent degradation secure the cap tightly upon storage. Avoid extended exposure to air.
CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany
Re-Blot is a trademark of Sigma-Aldrich Co. LLC
Sepharose is a registered trademark of Cytiva