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Protein Immunoblotting: (Western Blotting)

Western blotting is a technique used to detect specific proteins in a cell or tissue. It involves separating proteins by electrophoresis, transferring them to a membrane, and using antibodies to detect target proteins. The key steps are sample preparation, SDS-PAGE gel electrophoresis to separate proteins by size, transferring proteins to a membrane, and detecting target proteins using specific primary and secondary antibodies along with a visualization method. It allows identification and measurement of target proteins from complex mixtures.

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233 views19 pages

Protein Immunoblotting: (Western Blotting)

Western blotting is a technique used to detect specific proteins in a cell or tissue. It involves separating proteins by electrophoresis, transferring them to a membrane, and using antibodies to detect target proteins. The key steps are sample preparation, SDS-PAGE gel electrophoresis to separate proteins by size, transferring proteins to a membrane, and detecting target proteins using specific primary and secondary antibodies along with a visualization method. It allows identification and measurement of target proteins from complex mixtures.

Uploaded by

NANDA AHSANI
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Protein immunoblotting

(Western blotting)

Dr. Serageldeen A. A. Sultan


Lecturer of virology
Dept. of Microbiology
SVU, Qena, Egypt
[email protected]
Western blotting

-It is an analytical technique used to detect specific


proteins in a cell, tissue, organ, or body fluid. The
technique depends on the reaction of an antibody
with a protein that is immobilized on a thin
membrane.
-This technique can be used to identify a target
protein in a complex mixture, and to measure it’s
expression level.
-The method originated in the laboratory of George
Stark at Stanford
-The name Western blot was given to the technique
by W. Neal Burnette
-Southern blot?
-Northern blot?
-The transfer of DNA from agarose gel onto NC is
called Southern blot
- The transfer of RNA from agarose gel onto NC is
called Northern blot
Western blotting
-Identification of protein based on two distinguishing
properties:-
1- Molecular weight
2- Antibody binding specificity

Western blotting carried out through the following steps:-


1- Sample preparation
2- SDS-PAGE to separate native proteins
3- Transfer of protein to a membrane (nitrocellulose
or PVDF).
4- Detection of target proteins by specific antibodies
Sodium Dodecyl Sulfate-Polacrylamide Gel
Electrophoresis (SDS-PAGE)
- Proteins usually have a net positive or negative charge
(reflects the mixture of charged amino acids they contain)
-Protein will migrate at a rate that depends on its net
charge and on its size and shape
-In the mid-1960s SDS polyacrylamide-gel electrophoresis
(SDS-PAGE) was developed
-It uses a highly cross-linked gel of polyacrylamide as the
inert matrix through which the proteins migrate
-The gel is usually prepared immediately before use by
polymerization from monomers
-The pore size of the gel can be adjusted so that it is small
enough to retard the migration of the protein molecules of
interest
-The proteins in solution includes a powerful negatively
charged detergent, sodium dodecyl sulfate (SDS)
-SDS binds to hydrophobic regions of the protein
molecules, causing them to unfold into extended
polypeptide chain
-Mercaptoethanol (reducing agent) is usually added to break
any S - S linkages in the proteins so that all of the
constituent polypeptides in multi-subunit molecules can be
analyzed separately
Sample loading onto the SDS-PAGE gel
Sample preparation

SDS-PAGE
Large proteins are retarded much
more severely than small ones.
1-Loading samples onto SDS-PAGE gel

2- Electrophoresis

3-Staining
SDS-PAGE is a more powerful method of protein analysis
-Separate all types of proteins, including those that are
insoluble in water
- Membrane proteins, protein components of the
cytoskeleton, and that are part of large macromolecular can
all be resolved
- SDS-PAGE separates polypeptides according to size
(molecular weight and the subunit composition).
The transfer of the proteins onto a membrane

Why not add SDS in the transfer buffer?


Protein detection
1- Primary antibody incubation step.
The primary antibodies which specifically recognize the
proteins of intrest are used.

2- Secondary antibody incubation step.


Use of secondary antibody which recognizes the
primary antibody

3- Visualization step
Making the antigen-antibody complex visible (staining).
Colored product
(for easy detection)

Horseradish
peroxidase

Colorless Secondary
substrate antibody
(labeled)

Primary antibody
(antigen specific)

Protein mixtures are asborbed to an


inert surface

Protein detection in western blot


Protein detection
Western blotting
to detect a specific
antigen protein in
a protein mixture
using a specific
antibody.

Gel-separated antigen
proteins are transferred
onto a nitrocellulose
membrane before
being probed with
antibodies.

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