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
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

4. Blotting Techniques
Blotting
Techniques
Northern
Blotting
(RNA)
Eastern
Blotting
(PTM)
Southern
Blotting
(DNA)
Western
Blotting
(Protein)

5. 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

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
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.

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 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.

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.co
m/wiki/Eastern_blot

14. 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

15. 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

16. 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

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