The Western blot (Immunoblot), or western blotting, is a widely used analytical technique in molecular biology and immunogenetics to detect specific proteins in a sample of tissue homogenate or extract. Besides detecting the proteins, this technique is also utilized to visualize, distinguish and quantify the different proteins in a complicated protein combination. This slide share is meant to explain the principle, process and some minute details but the best way to understand any analytical technique is by performing it labs and doing several troubleshooting.

1. Western
Blotting
Ankit Mehra
PC/2022/202
Pharmacology & toxicology

2. INTRODUCTION
Western blotting is an important
technique in molecular biology, it
enables the separation and
identification of proteins from a
complex mixture of proteins.
Separation by size of
proteins
Transfer of protein to
solid support
Detecting target
protein using
antibodies
2
Typical western blot technique consist of following steps :
1. 2. 3.

3. Principle
A complex mixture of proteins is
separated based on molecular
weight through gel electrophoresis.
The separated proteins are then
transferred to membrane producing
band for each protein. The
membrane is then incubated with
labels of antibodies specific to the
protein of interest.
Separation of proteins
SDS-PAGE is used for
separation and it gives
uniform negative charge to
proteins
Transfer
membrane
Nitrocellulose (NC) or
Polyvinyl difluoride (PVDF) is
used
Immunodetection of
protein bands
Separated bands appear
according to their molecular
weight and are compared
with the protein ladder
3

4. Procedure :
4
Gel preparation- Stacking gel and separating gel.After preparation assemble the rack and add separating gel.
Overlay seperating gel with stacking gel and insert comb and wait for solidification.
Electrophoresis- Pour running buffer, add sample and molecular marker to gel, after removing comb. sample
running through the stacking gel and reaching the gel’s bottom according to their molecular weight.
Blotting-Transfer of separated proteins to the membrane by one of the two methods i.e by applying weight
to membrane or by using electroblotting techniques.
Blocking- Prevent non-specific binding of antibodies to the membrane.The most commonly used blotting
agent is 3-5 % BSA or non-fat dried milk in PBS.
Antibody probing- Primary antibody is added to the membrane.
Washing-Wash to remove extra unattached antibodies to the membrane.
Detection- After adding secondary antibody, substrate is passed that shows fluorescence.

5. Composition and importance of ingredients :
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A). Separating gel :
 Distilled water
 30% acrylamide – acts as a sieve through which proteins move in response to electric field.
 Seperating buffer – buffers for the separation of different protein bands.
 10% SDS- denatures and linearizes proteins, coating them with a negative charge.
 30% APS,TEMED –ammonium persulphate &TEMED catalyse the polymerization of acrylamide
solution into gel matrices.
B). Stacking gel :
 Distilled water
 30% acrylamide
 Stacking buffer
 10% SDS
 30% Aps,TEMED (but having a different concentration from separating gel).
Stacking buffer has a low pH-6.8 and Separating buffer has a high pH-8.8 because
glycine has an isoelectric point close to 6.8

6. Procedure :
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Assembled rack for gel
solidification
Add gel solution using a
transfer pipette
Add running buffer to the
electroporator
Add samples and molecular
marker to the gel,
removing the combs
(a) Samples running through the stacking gel (lower voltage).
(b): Samples running through the separating gel (higher
voltage).
Run the gel to the bottom
of the electroporator
Membrane after transfer

7. 7
Adding primary antibody
Final incubation and wait for some
time to show colour bands
Adding secondary antibody
Incubate after adding substrate
Protein bands are compared
with standard protein ladder

8. 8
RESULTS :

9. 9