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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...

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SouravBio
This article writter by SouravBio on May 23, 2021

Writer and Founder of Microbiologynote.com. I am from India and my main purpose is to provide you a strong understanding of Microbiology.

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Buffer preparation
Buffer preparation

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.
Eastern blotting flowchart
Eastern blotting flowchart

What is Far-estern Blotting?

  • The Far-Eastern blot is for the detection of lipid-linked oligosaccharides. HPTLC is first used to separate and then these are transferred to a blotting matrix before the oligosaccharides are detected by a specific binding protein (i.e. antibodies or lectins).
  • Far-estern Blotting technique was developed by Taki and colleagues in 1994.

Eastern blot Application

  • Used to detect fingerprints.
  • To detect protein modification. For example, detect the protein modifications in two bacterial species Ehrlichia- E. muris and IOE.
  • Used for binding studies by using various ligands.
  • Used to purify various phospholipids.
  • Help in translocation studies across biological membranes. 
  • Expression of post-translated proteins in several diseases.
  • To study Protein modifications in bacterial species.
  • Comparing the protein modification of two bacterial species.
  • Antigenic proteins of the non-virulent E. muris are more post-translationally modified than the highly virulent IOE.

References

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Writer and Founder of Microbiologynote.com. I am from India and my main purpose is to provide you a strong understanding of Microbiology.

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