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Eastern blotting Technique

Eastern blotting is a technique used to detect post-translational modifications (PTMs) of proteins, especially carbohydrate epitopes. It involves separating proteins by gel electrophoresis, transferring them to a membrane, then using probes like antibodies to detect PTMs. It is an extension of western blotting developed in 1979 by Towbin. The method involves separation, transfer to a membrane, addition of primary then secondary antibodies, and confirmation of the molecule of interest via autoradiography. It can detect PTMs in disease studies and compare modifications between bacterial species.

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Eastern Blotting Course: Advanced Research Techniques Course Code: BCH-512 Instructor: Dr. Muddassar Zafar Presented By: Zoqia Tariq (19121760-009)
Index • Blotting • Blotting Types • Eastern Blot • Method • Far Eastern Blotting • Applications • Trick • References
Blotting In Molecular Biology and Genetics, it is a method of transferring proteins, DNA or RNA onto a carrier membrane. After Gel Electrophoresis, transferring the sample from the gel onto the blotting membrane. After the Blotting, the transferred proteins, DNA or RNA are then visualized by color staining
Blotting Techniques Blotting Techniques Northern Blotting (RNA) Eastern Blotting (PTM) Southern Blotting (DNA) Western Blotting (Protein)
Eastern Blotting An Extension of the Western Blotting Analyze protein post translational modification (PTM) For Instance, To detect carbohydrate Epitopes Developed by Towbin in 1979
Definition of Eastern Blotting “A Molecular Biology Technique which is used for the identification of Post Translation Modification mostly carbohydrate Epitopes by transferring onto a membrane and analyzed using a probe.”
Method 1. Separation: targeted molecules are vertically separated by using gel electrophoresis 2. Transfer: Separated molecules are transferred horizontally on the nitrocellulose membrane 3. Addition of Primary Antibody: primary antibody is added to the solution. 4. Washing: Then wash it to remove unbound primary antibody 5. Secondary Antibody Addition: Add labeled secondary antibody with an enzyme which will emit the fluorescence when product is formed. 6. Confirmation: These labeled probes confirm the molecule of interest by autoradiography.
http://www.web- books.com/MoBio/Free/Ch9D.htm General Principle for Performing Eastern Blotting
Attachment of Antibodies with target molecule and luminescence Production
Example Fingerprint by Eastern Blotting: • Solasodine glycosides were developed on a TLC plate. • The TLC plate was covered with the PVDF membrane and blotted by short heating. • The blotted PVDF membrane was dipped in water containing NaIO4under stirring at room temperature for1 hr. • After washing with water, a carbonate buffer containingBSA was added and stirred for 3 hr. • ThePVDF membrane was washed twice with phosphate bufferfor 5 min • The PVDF membrane was immersed in anti-solamargine MAb, stirred at room temperature for 1 hr. After washing the PVDF membrane twice with phosphate buffer and water, a 1000-fold dilution of peroxidase-labeled goat anti-mouse IgG in phosphate buffer (pH 7.2) was added and stirred at room temperature for 1 hr. • The PVDF membrane was washed twice with phosphate buffer and water, then exposed to 4-choloro-1-naphthol (1 mg/mL)—H2O2(0.03%) in phosphate buffer(pH 7.2), and eastern blotting was stopped by washing with water. • The immuno-stained PVDF membrane was allowed to dry.
Animation
Far-Eastern Blotting • Technique developed in 1994 by Taki and colleagues Definition: 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)
https://wikivisually.co m/wiki/Eastern_blot
Flow Sheet Diagram Gel Electrophoresis (Separation) Transfer to Nitrocellulose membrane Addition of Primary Antibody Washing Step Addition of Secondary Antibody Confirmation of molecule of Interest
Applications Detection of protein modification. 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
Applications To study Protein modifications in bacterial species Comparing the protein modification of two bacterial species Antigenic proteins of the non-virulent E. muris is more post-translationally modified than the highly virulent IOE
Any Question? “I love to ask and have Questions, as I better learn from Questions; either by me or from me.”
References • Manu Tomar*Department of Biotechnology, Abdul KalamTechnical University, Lucknow, India (Introduction) • Tanaka, H., Putalun, W., & Shoyama, Y. (2012). Fingerprinting of Natural Product by Eastern Blotting Using Monoclonal Antibodies. Chromatography Research International, 2012. (Example) • https://wikivisually.com/wiki/Eastern_blot