Western Blotting

A. Aims:

In this lab session, you will observe and analyze the gel that was subjected to staining
and you will assess the efficiency of protein transfer onto a membrane, which were
performed following separation of your protein samples by sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) last week. You will subsequently
subject the membrane to Western blotting (also referred to as immunoblotting) in
order to detect a protein of interest in your samples.

B. Learning Objectives:

1. Observing and analyzing the gel that was stained with Coomassie Brilliant Blue
R-250 after separation of protein samples by SDS-PAGE last week
2. Assessing the efficiency of protein transfer onto a polyvinylidene difluoride
(PVDF) membrane that was performed following separation of protein samples by
SDS-PAGE last week
3. Understanding the concepts of Western blotting and the detection methods it
involves
4. Acquiring the ability to perform Western blotting
5. Using Western blotting to detect glyceraldehyde 3-phosphate dehydrogenase
(GAPDH) in protein samples that were extracted from MDA-MB-231 cells,
separated by SDS-PAGE and transferred onto a PVDF membrane
6. Monitoring equal protein loading by immunodetection of GAPDH

C. Introduction:

Western blotting is an analytical technique used for the detection of specific proteins
in samples extracted from whole tissues or from cells in culture. It utilizes gel
electrophoresis to separate native or denatured proteins, which are subsequently
transferred onto a membrane and stained with antibodies specific to the protein of
interest (refer to previous lab sessions for more information about protein extraction,
separation and transfer).

Western blotting is widely used in molecular biology, biochemistry and

immunogenetics, as well as other fields, where it allows comparing the expression
levels of a target protein. In addition, it has medical applications, such as those
involving disease diagnoses. For instance, HIV confirmatory test employs Western
blotting, whereby anti-HIV antibodies are detected in human serum samples.

Following protein transfer, blocking is performed due to the ability of membranes to

bind proteins, including antibodies, non-specifically. This is done in order to block
free sites on the membrane that remain unbound by transferred proteins. Blocking is
achieved by placing the membrane in a buffered saline solution of a protein, typically
bovine serum albumin (BSA) or non-fat dry milk in tris-buffered saline (TBS), and a
detergent, such as Tween 20 or Triton X-100. The protein binds to all sites on the
membrane where no proteins have been transferred. Consequently, non-specific
binding of the antibodies to the membrane would be prevented, thereby reducing
background signal.
After blocking, the membrane is probed for the protein of interest using specific
antibodies. Monoclonal or polyclonal antibodies may be used. Monoclonal antibodies
have monovalent affinity, meaning that they bind to one epitope on the antigen. Such
antibodies are secreted by an isolated B cell lineage, unlike polyclonal antibodies
which are derived from several B cell lineages within the body of the host species, i.e.
purified from serum, and are thus a collection of immunoglobulin molecules that
target a specific antigen, each recognizing a different epitope.

Antibodies are diluted in a buffered saline solution containing detergent and protein,
typically non-fat dry milk (or BSA) and Tween 20 (or Triton X-100) dissolved in
TBS. A buffered saline solution containing detergent and referred to as wash buffer,
typically Tween 20 or Triton X-100 dissolved in TBS, is used to remove the excess
unbound antibodies. The detection process traditionally involves two steps and is
achieved using a primary antibody specific to the target protein, followed by an
enzyme-conjugated secondary antibody specific to the primary antibody. The enzyme,
most commonly horseradish peroxidase (HRP), catalyzes the conversion of luminol, a
chemiluminescent substrate, in the presence of hydrogen peroxide as an oxidizing
agent into a luminescent product in proportion to the amount (or expression levels) of
the target protein. A photographic film is then exposed to the membrane and
developed in order to detect luminescence. This results in the production of dark
bands whose thickness is proportional to the amount of luminescent product and is
thus indicative of the protein expression levels (the thicker the band is the higher is
the expression level of the protein). A digital image may be captured by a special
camera instead. Such detection method is referred to as chemiluminescent detection
and is the most commonly used method in Western blotting. The results can be
analyzed either visually or by densitometry, which evaluates the relative amounts of
protein staining and quantifies the results in terms of optical density. Alternatively,
colorimetric detection involves enzyme-catalyzed conversion of a chromogenic
substrate into an insoluble colored reaction product that precipitates next to the
enzyme and can be visualized on the membrane and photographed. Another method
of detection involves the use of a fluorophore-linked secondary antibody and is
referred to as fluorescent detection. Upon excitation, the fluorescent dye produces a
static light, which allows for precise and accurate detection of the target protein. A
special camera is used to capture a digital image of the Western blot. On the other
hand, radioactive detection involves the use of a radioactively-labeled secondary
antibody, and is followed by the exposure of a medical X-ray film to the membrane
and development, as in chemiluminescent detection. Since other methods are safer,
cheaper and faster, radioactive detection is rarely used.

Membranes can be used for secondary probing. This involves stripping antibodies off
and reusing the membrane for subsequent probing. PVDF membranes, unlike
nitrocellulose membranes, can be stripped several times in order to optimize detection
of the target protein (i.e. antibody concentrations) or to detect other proteins.

D. Experimental Procedure:

In this experiment, you will observe and analyze the gel that was stained with
Coomassie Brilliant Blue R-250 and assess the efficiency of protein transfer onto a
PVDF membrane, which were performed following separation of your protein
samples by SDS-PAGE last week. You will subsequently perform Western blotting
on the membrane to detect GAPDH in your protein samples, and you will use the
results to monitor equal protein loading.

 Remove the membrane from its storage at 4 °C.

 Reactivate the membrane as follows:

 10 seconds in methanol
 5-10 minutes in double distilled water (DDW)

 Place the membrane in a box and block in 50 ml of blocking buffer (TBS with 5%
non-fat dry milk and 0.1% Tween 20) at 37 °C. This is done on a shaker for 30
minutes.
 Meanwhile, prepare the primary antibody (goat anti-GAPDH antibody). This is
done by diluting the antibody in a ratio of 1:200 in antibody buffer (TBS with 1%
non-fat dry milk and 0.1% Tween 20), i.e. by adding 10 µl of the antibody to 2 ml
of the antibody buffer.
 After blocking, remove the membrane and place in a plastic bag. Add the prepared
antibody to the bag, and incubate the membrane at 37 °C for 1 hour on a shaker.
 After the primary antibody, remove the membrane, place it in a box and wash with
about 50 ml of wash buffer (TBS with 0.1% Tween 20). This is done at room
temperature for 5 minutes on a shaker.
 Repeat the previous step two more times.
 Meanwhile, prepare the secondary antibody (mouse anti-goat IgG-HRP). This is
done by diluting the antibody in a ratio of 1:5,000 in antibody buffer (TBS with
1% non-fat dry milk and 0.1% Tween 20), i.e. by adding 1 µl of the antibody to 5
ml of the antibody buffer.
 After washing, remove the membrane and place in a box containing the prepared
antibody. Incubate the membrane at 37 °C for 30 minutes on a shaker.
 After the secondary antibody, remove the membrane, place it in a box and wash
with about 50 ml of wash buffer (TBS with 0.1% Tween 20). This is done at room
temperature for 5 minutes on a shaker.
 Repeat the previous step two more times.
 Meanwhile, clean the plastic covers of the cassette with 70% ethanol, and switch
on the XOMAT machine.
 At the end of the last wash, prepare the substrate solution under dim room light,
since it is light sensitive. This is done by mixing equal volumes of reagents A and
B (i.e. 0.5 ml from each) in a tube wrapped with aluminum foil.
 After washing, remove the membrane, air dry and place between the plastic covers
of the cassette.
 Drip the prepared substrate solution onto the entire surface of the membrane, and
leave for 1-2 minutes.
 Close the plastic cover of the cassette, and remove the air bubbles.
 Close the cassette and take along with the box of X-ray films and scissors to the
XOMAT room in order to expose and develop the films.
 Turn the regular light off, and work under red light. Cut an X-ray film at one
corner in a similar manner to the membrane, and expose for 1 minute. This is done
by placing the film on the plastic cover above the membrane and closing the
cassette.
 Remove the film and insert into the XOMAT machine. After about 2 minutes, the
developed film will come out of the machine.
 Securely close the box of X-ray films, and turn the regular light on and the
XOMAT machine off.
 Remove the membrane from the cassette, wash in wash buffer for a few minutes,
and then place in a filter paper and store at 4 °C for later reprobing if need be.
 Analyze the results.