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Eastern blot Protocol, Principle, Application

A Molecular Biology Technique is used for the identification of Post Translation Modification mostly carbohydrate Epitopes by transferring onto a membrane and analyzed using a probe.

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MN Editors

What is Eastern blotting?

A Molecular Biology Technique is used for the identification of Post Translation Modification mostly carbohydrate Epitopes by transferring onto a membrane and analyzed using a probe.

  • It is an extension of the western blotting technique which is developed by Towbin in 1979.
  • Eastern blotting technique is used to examine protein post-translational modifications including the addition of lipids, phosphates, and glycoconjugates. 
  • Sometimes it also used to detect carbohydrate epitopes. 
  • In this method, the prospo protein is blotted from sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel onto a polyvinylidene fluoride or nitrocellulose membrane. After that, the proteins are studied for post-translational modifications with the help of probes that may detect lipids, carbohydrates, phosphorylation, or any other protein modification.

Aim

To analyze protein post-translational modifications.

Eastern blot Principle

In the eastern blotting technique the protein samples are first separated by usign gel electrophoresis then are transferred onto a nitrocellulose membrane. After that, the primary antibodies are added, these will bind with the nitrocellulose membrane. Then the unbounded primary antibodies are removed by washing the membrane. After that, the enzyme-labeled secondary antibodies are added which will binds with the primary antibody.

Eastern blot Procedure

  1. Separation: The supplied sample or test sample is vertically separated by using gel electrophoresis.
  2. Transfer: After separation, the molecules are horizontally transferred onto a nitrocellulose membrane.
  3. Addition of Primary Antibody: After completion of transfer the primary antibodies are added to the solution.
  4. Washing: Wash the membrane to remove the unbounded primary antibody.
  5. Addition of Secondary antibody: Add the enzyme-labeled secondary antibody which will emit the fluorescence when the product is formed.
  6. Autoradiography: Perform autoradiography to confirm the molecule of interest by labeled probes.

What is Far-estern Blotting?

Eastern blot Application

References

Citation

APA

MN Editors. (May 23, 2021).Eastern blot Protocol, Principle, Application. Retrieved from https://microbiologynote.com/eastern-blotting-technique/

MLA

MN Editors. "Eastern blot Protocol, Principle, Application." Microbiology Note, Microbiologynote.com, May 23, 2021.

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