A secondary antibody is used to

detect the primary antibody that is

bound to the target protein.
Secondary antibodies are
conjugated to enzymes or labeled
with fluorescence to be easily seen
Secondary Antibody by detection systems.

Reactions from the enzyme-tagged

secondary antibody as well as the
fluorescence produced is detected
using an imaging system. The signal
detected is proportional to the
amount of protein present.
Detection

For more information visit:

ThermoFisher Western Blotting Handbook
https://tools.thermofisher.com/content/sfs/
brochures/1602761-Western-Blotting-
Handbook.pdf

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 Proteins are commonly separated
Western blot or also known as immunoblot is an using polyacrylamide gel
electrophoresis (PAGE) to
analytical technique of detecting specific proteins in
characterize individual proteins in
a sample of tissue homogenate or extract. It was a complex sample or to examine
first developed by the group of Harry Towbin in 1979 multiple proteins within a single
as a modified version of Southern blot. Gel Electrophoresis sample.

It is now a widely-accepted technique for protein

analysis and is used to demonstrate antibody
Both polyacrylamide gel and
specificity, confirm gene expression, detect post- nitrocellulose are "sandwiched"
translational modifications in proteins and diagnose between two electrodes
submerged in a conducting
diseases like HIV/AIDS. This method is, however, solution. When an electric field is
dependent on the use of a high-quality antibody applied, the proteins move out of
the polyacrylamide gel and onto
directed against a desired protein to achieve an
the surface of the membrane
accurate result. where it is tightly attached. Membrane Transfer

It is important to block the

remaining surface of the
membrane to prevent nonspecific
binding of the detection antibodies
during subsequent steps. A variety
of blocking buffers ranging from
milk to normal serum to highly
Blocking purified proteins have been used to
block free sites on a membrane.

Primary antibodies which

HIV/AIDS
Used as a confirmatory test for HIV after
screening using ELISA
CREUTZFELDT-JAKOB DISEASE
A prion disease associated with eating cattle that
are infected with Mad Cow Disease
LYME DISEASE
A disease caused by Borrelia spp. often
transmitted to humans by tick bites
HEPATITIS B
Sometimes used as a confirmatory test for
Hepatitis B together with HBsAg
The “Blotting Compass”
Did you know that it was in 1970, Southern blot was first
developed? It was named after its inventor, Edwin
Southern which is a method used to detect DNA
sequences. Two years later, another blotting technique
that now detects RNA was invented and was
humorously named as northern blot, following the
pioneer of Southern blot.
Over time, other blotting methods like western blot—
which detects protein; eastern blot—which detects
carbohydrates; and southwestern blot—which detects
DNA-binding protein; are just a wordplay to the
eponymously-named Southern blot.
recognizes the specific protein are
added to the blocked membrane.
Often times, the primary antibody
is not directly detectable and are
therefore tagged with a secondary
antibody or other detection
reagents.
Washing
Primary Antibody
Washing steps are necessary to
remove unbound reagents and
reduce background, increasing the
signal:noise ratio. Insufficient
washing will allow high background,
while excessive washing may result
in decreased sensitivity caused by
elution of the antibody and/or
antigen from the blot.