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technique(s)
western blot: suitable
detection method
chemiluminescent
General description
Western blotting is a commonly used technique for studying protein function and localization. Typically, protein samples are electro-phoresed 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 re-probing 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) Re-analysis 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 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 Western Blot Recycling Kit include:
·*No pungent-smelling b-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 10 minutes at room temperature.
·*Reblocking of blots may be avoided in most instances.
·*Blots may be reused in less than 15 minutes.
Stripping and re-probing 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) Re-analysis 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 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 Western Blot Recycling Kit include:
·*No pungent-smelling b-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 10 minutes at room temperature.
·*Reblocking of blots may be avoided in most instances.
·*Blots may be reused in less than 15 minutes.
Application
The MILLIPORE Re-Blot 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 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.
The Re-Blot 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.
Components
Antibody Stripping Solution (10x) - (1 container, 50 mL). Blocking Buffer (20x) milk-based - (1 container, 50 mL).
Storage and Stability
Kit components should be stored at 2-8° 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.
Disclaimer
Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
Storage Class Code
10 - Combustible liquids
Certificates of Analysis (COA)
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