.

Western blotting & SDS-

PAGE
 Western Blot

• Western blots allow investigators to

determine the molecular weight of a
protein and to measure relative amounts
of the protein present in different samples.
 Applications & Advantages
Applications:
To determine the molecular weight of a
protein (identification)
To measure relative amounts (quantitation)
of the protein present in complex mixtures of
proteins that are not radiolabeled (unlike
immunoprecipitation)
Advantages:
WB is highly sensitive technique
As little as 1-5 ng of an average-sized protein can
be detected by WB
 …Western Blot
• Proteins are separated by gel
electrophoresis, usually SDS-PAGE.
• The proteins are transferred to a sheet of
special blotting paper called nitrocellulose.
• The proteins retain the same pattern of
separation they had on the gel.
 ..Western Blot
• The blot is incubated with a generic
protein (such as milk proteins) to bind to
any remaining sticky places on the
nitrocellulose.
• An antibody is then added to the solution
which is able to bind to its specific protein.
• The antibody has an enzyme (e.g.
alkaline phosphatase or horseradish
peroxidase) or dye attached to it which
cannot be seen at this time.
 ..Western Blot
• The location of the antibody is revealed
by incubating it with a colorless substrate
that the attached enzyme converts to a
colored product that can be seen and
photographed.
..Western Blot
 SDS-PAGE (PolyAcrylamide
Gel Electrophoresis)
• SDS-PAGE, sodium dodecyl sulfate
polyacrylamide gel electrophoresis, is a
technique widely used in biochemistry,
forensics, genetics and molecular biology:
• to separate proteins according to their
electrophoretic mobility (a function of length of
polypeptide chain or molecular weight).
• to separate proteins according to their size, and
no other physical feature.
 …SDS-PAGE

• SDS (sodium dodecyl sulfate) is a

detergent (soap) that can dissolve
hydrophobic molecules but also has a
negative charge (sulfATE) attached to it.
Fig.1Before SDS: Protein (pink line) incubated with the denaturing detergent SDS showing negative
and positive charges due to the charged R-groups in the protein.
The large H's represent hydrophobic domains where nonpolar R-groups have collected in an attempt
to get away from the polar water that surrounds the protein.
After SDS: SDS disrupt hydrophobic areas (H's) and coat proteins with many negative charges which
overwhelms any positive charges the protein had due to positively charged R-groups.
The resulting protein has been denatured by SDS (reduced to its primary structure-aminoacid
sequence) and as a result has been linearized.
 ..SDS
• SDS (the detergent soap) breaks up
hydrophobic areas and coats proteins with
negative charges thus overwhelming
positive charges in the protein.
• The detergent binds to hydrophobic
regions in a constant ratio of about 1.4 g of
SDS per gram of protein.
 ..SDS
• Therefore, if a cell is incubated with SDS,
the membranes will be dissolved, all the
proteins will be solubalized by the
detergent and all the proteins will be
covered with many negative charges.
 PAGE
•
If the proteins are denatured and put into
an electric field (only), they will all move
towards the positive pole at the same rate,
with no separation by size.
• However, if the proteins are put into an
environment that will allow different sized
proteins to move at different rates.
• The environment is polyacrylamide.
• the entire process is called
polyacrylamide gel electrophoresis
 ..PAGE
• Small molecules move through the
polyacrylamide forest faster than big
molecules.
• Big molecules stays near the well.
 …SDS-PAGE
• The end result of SDS- PAGE has two
important features:
1) all proteins contain only primary structure
&
2) all proteins have a large negative charge
which means they will all migrate towards
the positive pole when placed in an
electric field.
The actual bands are equal in size, but the
proteins within each band are of different
sizes.
Sample of SDS- PAGE
Protein gel (SDS-PAGE) that has
been stained with Coomassie
Blue.
 What happens after
electrophoresis?
• 1. Fix the proteins in the gel and then stain
them.
• 2. Electrophorectic transfer to a
membrane and then probe with
antibodies- (Western blotting)
 ..Western blotting
• Western blot analysis can detect one protein in
a mixture of any number of proteins while giving
you information about the size of the protein.
• This method is, however, dependent on the use
of a high-quality antibody directed against a
desired protein.
• This antibody is used as a probe to detect the
protein of interest.
 Western Blot followed by SDS
• Proteins are separated using SDS-polyacrylamide gel
electrophoresis which separates proteins by size.
• Nitrocellulose membrane is placed on the gel. The
actual blotting process may be active (electroblotting) or
passive (capillary).
• Electroblotter is used for faster and more efficient
transfer of protein from gel to membrane
• Sandwich of filter paper, gel, membrane and more filter
paper is prepared in a cassette, which is placed between
platinum electrodes.
• An electric current is passed through the gel causing the
proteins to electrophorese out of the gel and onto the
nitorcellulose membrane.
 Western blotting

The main steps of blotting technique in

a chronological order will be as follows:
• Blocking
• Probing with the specific antibody(ies)
• Wash
• Detection
• Washing
• X-ray (Gel Documentation System)
 WB Procedure; Briefly…
2 1

3 4

www.bio.davidson.edu/.../method/Westernblot.html
Direct Detection Method
Indirect Detection Method
WB: examples of used
substrates
Chemiluminescent substrates
Enhanced ChemiFluoresenct (ECF)
WB Detection
Immunoprecipitation :The combined procedures of immunoprecipitation
and SDS-PAGE can be a powerful tool to assess the amount and size of
an antibody-reactive antigen present in a complex protein mixture. The
basic protocol uses a primary antibody followed by a secondary
antibody-agarose conjugate to immunoprecipitate the antigen.
Immunoprecipitation Protocol

Wash adherent cells twice in the dish or flask with ice-cold PBS and drain off PBS. Wash non-
adherent cells in PBS and centrifuge at 800 to 1000 rpm in a table-top centrifuge for 5 minutes to
pellet the cells.
Add ice-cold modified RIPA buffer to cells (1 ml per 10 7 cells/100 mm dish/150 cm2 flask; 0.5
ml per 5 x 106 cells/60 mm dish/75 cm2 flask).
Scrape adherent cells off the dish or flask with either a rubber policeman or a plastic cell scraper
that has been cooled in ice-cold distilled water. Transfer the cell suspension into a centrifuge
tube.
Gently rock the suspension on either a rocker or an orbital shaker at 4 degrees Celsius for 15
minutes to lyse cells.
Centrifuge the lysate at 14,000 x g in a precooled centrifuge for 15 minutes. Immediately transfer
the supernatant to a fresh centrifuge tube and discard the pellet.
To prepare protein A or G agarose/sepharose, wash the beads twice with PBS and restore to a
50% slurry with PBS. It is recommended to cut the tip off of the pipette when manipulating
agarose beads to avoid disruption of the beads.
Pre-clear the cell lysate by adding 100 ul of either protein A or G agarose/sepharose bead slurry
(50%) per 1 ml of cell lysate and incubating at 4 degrees Celsius for 10 minutes on a rocker or
orbital shaker. Pre-clearing the lysate will reduce non- specific binding of proteins to the agarose
or sepharose when it is used later on in the assay.
Remove the protein A or G beads by centrifugation at 14,000 x g at 4 degrees Celsius for 10
minutes. Transfer the supernatant to a fresh centrifuge tube.
Determine the protein concentration of the cell lysate, e.g. by performing a Bradford
assay. Dilute the cell lysate at least 1:10 before determining the protein concentration
because of the interference of the detergents in the lysis buffer with the Coomassie-
based reagent.
Dilute the cell lysate to approximately 1 ug/ul total cell protein with PBS to reduce the
concentration of the detergents in the buffer. A more concentrated cell lysate (i.e., 10
ug/ul) may be necessary to immunoprecipitate a protein, which is found in low levels in a
cell model. Add the recommended volume of the immunoprecipitating antibody (see
datasheet for detailed information) to 500 ul (i.e., 500 ug) of cell lysate. The optimal
amount of antibody that will quantitatively immunoprecipitate the protein of interest
should be empirically determined for each cell model.
Gently rock the cell lysate/antibody mixture for either 2 hours or overnight at 4 degrees
Celsius on a rocker or an orbital shaker. A 2 hour incubation time is recommended for
the immunopecipitation of active enzymes for kinase or phosphatase assays.
Capture the immunocomplex by adding 100 ul protein A or G agarose/sepharose bead
slurry (50 ul packed beads) and gently rocking on either a rocker or orbital shaker for
either 1 hour or overnight at 4 degrees Celsius. In many instances, immunocomplex
capture can be enhanced by adding 2 ug of a bridging antibody (e.g., rabbit-anti-mouse
IgG). This is especially important with antibodies, which bind poorly to protein A, such as
mouse IgG1 or antibodies generated in chicken.
Collect the agarose/sepharose beads by pulse centrifugation (i.e., 5 seconds in the
microcentrifuge at 14,000 rpm). Discard the supernatant and wash the beads 3 times
with 800 ul ice-cold modified RIPA buffer. Occasionally, washing with modified
RIPA buffer will strip the immunocomplex off of the agarose/sepharose beads. In
these cases, washing with the milder PBS is recommended.
Resuspend the agarose/sepharose beads in 60 ul 2x sample buffer and mix gently.
This will allow for sufficient volume to run three lanes. The agarose/sepharose
beads are boiled for 5 minutes to dissociate the immunocomplexes from the beads.
The beads are collected by centrifugation and SDS-PAGE is performed with the
supernatant. Alternatively, the supernatant can be transferred to a fresh
microcentrifuge tube and stored frozen at –20 degrees Celsius for later use. Frozen
supernatant should be reboiled for 5 minutes directly prior to loading on a gel.
 Terminologies..
• The Western blot (alternatively, protein immunoblot) is
an analytical technique used to detect specific proteins in
a given sample of tissue homogenate or extract.
• A Southern blot is a method routinely used in molecular
biology for detection of a specific DNA sequence in DNA
samples.
• The northern blot is a technique used in molecular
biology research to study gene expression by detection
of RNA.
• Southwestern blotting, based along the lines of
Southern blotting (which was created by Edwin
Southern) and first described by B. Bowen and
colleagues in 1980, is a lab technique which involves
identifying and characterizing DNA-binding proteins
(proteins that bind to DNA).
 Terminologies..
• Dot blot a mixture containing the molecule
to be detected is applied directly on a
membrane as a dot.
• Protein detection using the dot blot
protocol is similar to western blotting in
that both methods allow for the
identification and analysis of proteins of
interest.