Western blotting is a technique used to detect specific proteins in a sample. It involves separating proteins by electrophoresis, transferring them to a membrane, and using antibodies to locate the target protein. The key steps are sample preparation, SDS-PAGE gel electrophoresis to separate proteins by size, transferring proteins from the gel to a membrane, blocking the membrane to prevent nonspecific antibody binding, probing the membrane with antibodies to detect the target protein, washing unbound antibodies, and detecting the bound antibodies to analyze and image the results. Western blotting allows identification and quantification of proteins in a given sample.

1. WESTERN BLOTTING

2. WESTERN BLOTTING
• The Western blot is an analytical technique
used to detect specific proteins in a given
sample of tissue homogenate or extract.
• Western blot for Proteins was Developed by
George Stark using antibodies to locate Proteins.

3. The Western blotting workflow
1. Sample preparation
2. SDS-PAGE
3. Transfer
4. Blocking
5. Antibody probing
6. Detection
7. Analysis

4. SAMPLE PREPARATION
• Protein from all sources can be used for western blotting
such as from single cell, whole tissues, extracellular matrices,
biological fluids.
• Protein can be extracted from the sample in three distinct
steps:
• Cell lysis: Physical method (ultra sonication, homogenization
etc.)
Chemical method (use of detergents)
Biological method (enzymatic digestion)
• Protein isolation from the rest of cellular components:
Centrifugation, DNase and other lysis buffer is used for the
removal of cell organelles, nucleic acids, polysaccharides etc.

5. SDS-PAGE
SDS-PAGE separates proteins based on their
molecular weight & all the separated proteins
contain a negative charge due to presence of
anionic detergent SDS.

6. SAMPLE LOADING

7. PROTEIN TRANSFER
Protein are transferred from the gel to a solid support
membrane such as nitrocellulose or polyvinylidene
difluoride (PVDF).
 The proteins transferred from the gels are immobilized
which makes it possible to detect the proteins on the
membrane using specific antibodies.
 Electro-blotting is mainly used which uses electric field
to pull proteins from the gel onto the PVDF or
nitrocellulose membrane.
 The proteins move from within the gel onto the
membrane while maintaining the organization they
had within the gel.

10. PROTEIN STAINING
After gel electrophoresis, it may be necessary to
confirm that all the proteins in the gel have been
completely eluted.
As proteins are not directly visible in the gel, the
gel must be stained.
Proteins are usually stained with dyes such as
coomassie blue, silver stain, or deep purple.

12. BLOCKING
 For meaningful results, the antibodies must bind
only to the protein of interest and not to the
membrane.
 Non-specific binding (NSB) of antibodies can be
reduced by blocking the unoccupied sites of
membrane with an inert protein or non-ionic
detergent.
 Blocking agents should possess greater affinity
towards membrane than the antibodies.

13. BLOCKING AGENTS
The most common blocking agents are:
a) Bovine serum albumin(BSA)
b) Non-fat milk
c) Casein
d) Gelatin
e) Dilute solution of Tween 20.

14. ANTIBODY PROBING
 Western blotting protocols usually uses a non-labeled
primary antibody directed against the target protein
and labeled secondary antibody directed against the
constant region of the primary Antibody.
 The secondary antibody serves not only as a carrier of
the label but is also a mechanism to amplify the
emitted signals because secondary antibodies are most
often polyclonal and can bind primary antibody at
different epitopes simultaneously.

15. WASHING
Unbound antibodies can cause high
background and poor detection.Hence
Washing the blot removes unbound
antibodies from the membrane.
A dilute solution of tween-20 in TBS or PBS
buffer is commonly used for washing.

16. PROTEIN DETECTION
 After the unbound probes are washed away,
the western blotting is now ready for detection
of the probes that are labeled and bound to the
protein of interest.
 Enzymes such as alkaline phosphatase(AP), &
Horse-radish peroxidase(HRP) are widely used
in detection of proteins.

17. ANALYSIS & IMAGING
This is the last & major step of the western blotting
technique.
Detection of signals, using either X-Ray film, scanners
or a CCD, results in one or more visible protein bands
on the membrane image.
The molecular weight of the protein can be estimated
by comparison with marker proteins and the amount
ofprotein can be determined as this is related to band
intensity.
Qualitative & quantitative analysis can be done in order
to verify the absence or presence of specific proteins
ofinterest.

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SHANKARNARAYAN A M
BLH6039