Western blotting

Steps of protein detection using Western

blotting technique
1- Vertical electrophoresis
2- Membrane blotting
3- Membrane blocking
4- Washing steps
5- Incubation with 1ry antibody overnight
6- Washing steps
7- Incubation with 2ry antibody for about 1h
8- Washing steps
9- Membrane development and signal detection
 Membrane blocking
Western blotting involves the immobilization of
biomolecules on a membrane via hydrophobic
interactions.
As non-specific binding of antibodies to the membrane
is detrimental to the specificity and sensitivity of the
assay, it is essential to "block" spaces not already occupied
by proteins.
Your choice of blocking strategy will be guided by your
samples and the antibodies used.
As a starting point for choosing blocking agents, buffers
and optimal conditions, begin by using those recommended
by the manufacturers of the detection reagents.
 Two main classes of blocking agents are commonly used
for Western blotting:
A- proteins
B- non-ionic detergents
 A- Proteins as blocking agents
They are known as permanent blocking agents as they
form a physical attachment to the membrane.
Requirements of blocking agents:
1- It is critical that the blocking agent has a greater affinity
for the membrane than the antibodies used in subsequent
steps.
2- the blocking agent should fill all unoccupied binding sites
without disrupting binding interactions between transferred
proteins and the membrane.
 The most common permanent blocking agents include
bovine serum albumin (BSA), non-fat milk, normal goat
Serum and casein
 B- Detergents as blocking agents

Detergents inhibit non-specific hydrophobic binding of

proteins to membranes.
They are considered non permanent blocking agents since
they do not attach to the Membrane and can be removed
in a simple washing step.
A solution of Tween-20 is commonly used, but
alternatives may be considered, such as Triton X-100,
sodium dodecyl sulfate (SDS) or NP40, which are especially
useful to counter strong background signals.
Detergent blocking agents are usually used in
conjunction with protein blocking agents.
 Antibody probing

Once your protein samples are separated and transferred

onto a membrane, the protein of interest is detected and
localized using a specific antibody.
Usually, Western blotting protocols utilize a non-labeled
primary antibody directed against the target protein and a
species-specific, labeled secondary antibody directed
against the constant region of the primary antibody.
 Primary antibody probing

Following the blocking step, the protein of interest can

be detected using antibodies.
Special considerations concerning 1ry antibodies:
1- If you purchase a commercially available primary
antibody, ensure that it has been validated for Western
blotting applications according to the manufacturer.
2- You can save money by reusing the primary antibody
solution, but be careful how you store it so that it retains
its activity.
3- Use clean tubes and store refrigerated when not in use
4- You can also consume less primary antibody by
incubating the membrane in a smaller box.
5- If the protein of interest is well characterized i.e. if you
know its molecular weight and where it is expected to
migrate on the membrane, cut the membrane to the
appropriate smaller size after transfer.
 Washing step
After primary antibody probing, it is necessary to wash
the membrane in order to remove excess antibody that
could cause high background and, consequently, a low
signal-to-noise ratio.
A low concentration detergent solution, such as 0.05 to
0.1% Tween-20 in PBS or TBS is commonly used, especially
after incubation with highly concentrated antibody
solutions or crude extracts.
PBS-Tween is suitable for most washing applications.
However, TBS-Tween is preferable where the target
proteins are phosphorylated, as the phosphate in PBS may
interfere with antibody binding.
2- Secondary antibodies

A wide variety of secondary antibodies are commercially

available and your choice will depend firstly on the species
in which the primary antibody was produced.
If, for example, the primary antibody was of the IgG
isotype and produced in goat, the secondary antibody must
be an anti-goat IgG antibody produced in another species.
The procedures for incubation of the secondary antibody
solution and the membrane are essentially similar to those
described for the primary antibody.
Role of secondary antibody:
1- secondary antibody serves as a carrier of the label.
2- is also a mechanism to amplify the emitted signals, as
many secondary antibodies can theoretically bind
simultaneously to the primary antibody.
For this reason, secondary antibodies are most often
polyclonal and can target epitopes on the framework
regions of the primary antibody; specificity is thus limited
to species and immunoglobulin isotype.
The signal emitted by the labeled secondary antibody is
then measured and is proportional to the quantity of
protein of interest present on the membrane.
Detection step
 Chemiluminescence

The HRP-conjugated secondary antibody binds to the

primary antibody, specifically bound to the target protein
on the membrane.
After the addition of a luminol peroxide detection
reagent, the HRP enzyme catalyzes the oxidation of
luminol in a multi-step reaction.
The reaction is accompanied by the emission of low
intensity light at 428 nm.
 In the presence of certain chemicals, the emitted light is
enhanced up to 1000-fold, making it easier to detect, and
thus increasing the sensitivity of the reaction in a process
known as enhanced chemiluminescence (ECL).
Several enhancers can be used, but the most effective
are modified phenols, especially p-iodophenol, which
increases HRP turnover rate and assists in the transfer of
electrons from luminol to the enzyme.
The intensity of signal is a result of the number of
reacting enzyme molecules and is thus proportional to the
amount of antibody, which is related in turn to the amount
of protein on the blot
The emitted signals can be detected using X ray films
Examples of signal detection using X ray films and ECL kit