Eastern blotting Technique

Eastern Blot (rare to find topic) is the very advanced technique to study the protwin

1. Eastern Blotting Course : Advanced Research Techniques Course Code : BCH-512 Instructor: Dr. Muddassar Zafar Presented By : Zoqia Tariq (19121760-009)

2. Index Blotting Blotting Types Eastern Blot Method Far Eastern Blotting Applications Trick References

3. Blotting

4. Blotting Techniques

5. Eastern Blotting

6. 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.”

7. Method Separation : targeted molecules are vertically separated by using gel electrophoresis Transfer: Separated molecules are transferred horizontally on the nitrocellulose membrane Addition of Primary Antibody : primary antibody is added to the solution. Washing: Then wash it to remove unbound primary antibody Secondary Antibody Addition : A dd labeled secondary antibody with an enzyme which will emit the fluorescence when product is formed. Confirmation : These labeled probes confirm the molecule of interest by autoradiography.

8. http://www.web-books.com/MoBio/Free/Ch9D.htm General Principle for Performing Eastern Blotting

9. Attachment of Antibodies with target molecule and luminescence Production

10. 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 shor t 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.

11. Animation

12. 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 )

13. https://wikivisually.com/wiki/Eastern_blot

14. Flow Sheet Diagram

15. Applications

16. Applications

17. Any Question? “I love to ask and have Questions, as I better learn from Questions; either by me or from me.”

18. 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