Technical analysis of proteins:

WESTERN BLOT

Introduction

 The Western blot is a protein transfer. It is a molecular biological method for the
detection and identification of specific proteins in a biological sample.

 It is a complementary diagnostic tool.

 This technique born of advances in proteomics, molecular biology and

Immunofluorescence.

Western Blot

Electrophoresis Transfer Revelation

Polyacrylamide gel
Nitrocellulose Detection of the
membrane protein

Molecular weight

Evaluation of size and

concentration ... etc.

Principal

 The Western blot is a laboratory test that can find in the blood serum proteins,
particularly antigenic viral proteins before antibodies became manifest.

 It also serves as a confirmation when the ELISA is positive, even partially. In this
case, it is the anti-HIV antibodies produced by patients who are wanted.
 * Preparation of
samples
The samples are rapidly cooled

homogenized by sonication

Lysed by pads high concentration of salts

homogenate of all compartments

Assay by the Bradford method

Denaturation of proteins that is boiled in a buffer solution

Preparation of samples

Boiling Denatures proteins by breaking the weak intramolecular bonds.

SDS Provides them with an environment rich in negative charges

β mercaptoethanol Prevents the regeneration of disulfide bonds

Glycerol Increases buoyancy.

* The gel electrophoresis
The electrophoresis is intended to separate charged molecules through a gel under the effect of an
electric field.

The polyacrylamide gel electrophoresis is used to:

determine the number of subunits of a protein and determine their respective molecular weight;

to assess the degree of purification of a protein;

and separate proteins reveal for the Western blot (reaction with one or more antibodies)

* The polyacrylamide gel

electrophoresis
The network of polyacrylamide is formed by polymerizing acrylamide monomer in the presence of
small amount of bis acrylamide

bis acrylamide acrylamide = 2 monomers linked by a methyl group

 * The polyacrylamide gel
electrophoresis
The polymerization of acrylamide is an example of catalysis by free radicals initiated by the addition
of ammonium persulfate and TEMED.

TEMED catalyzes the decomposition of persulfate ions to give a free radical R • (a molecule with one
electron):

* The polyacrylamide gel

electrophoresis
If this represents a radical R and M acrylamide monomer, we can represent the polymerization as
well:
 * The polyacrylamide gel
electrophoresis

a, b …j represents the
number of acryl
monomers. Plud they
are extras the gel is
crosslinked
 * The polyacrylamide gel
electrophoresis
The system used for Western blot two successive modes of electrophoretic migration:

an isotachophoresis (4% stacking gel);

then a single-phase zone electrophoresis (separating gel 7-15%).

The portion of each other is done with a change in pH and an increase in concentration of acrylamide
gel.
 * The polyacrylamide gel
electrophoresis
The proteins have an electrophoretic migration inversely proportional to the logarithm of their
molecular mass.

* The polyacrylamide gel

electrophoresis
The sample buffer poured into each well contains a dye ionized, usually bromophenol blue that
tracks electrophoresis.

direction of
electrophoresi
s
* The polyacrylamide gel
electrophoresis
 * The polyacrylamide gel
electrophoresis
The gel concentration is intended to concentrate the deposit made in the well in a thin strip of
proteins.

The isotachophoresis uses the fact that the SDS-protein complex has an electrophoretic mobility
intermediate between glycinate ions (buffer tank) and chloride ions (in the gel).

As the different SDS-protein complexes pass through a separating gel, with relatively small pores, they
split due to the molecular sieve properties of the gel.

* The polyacrylamide gel

electrophoresis
 * The polyacrylamide gel
electrophoresis

Bromophenol blue, contained in the sample buffer, is absolutely not retarded and therefore
indicates the electrophoretic migration front.

When the dye reaches the bottom of the gel, electrophoresis is stopped.

* The polyacrylamide gel

electrophoresis
The patient's Placed in an electric
serum is placed field, proteins migrate
on one edge of the as a function of their
plate SDS-PAGE molar mass

High Low
molecula molecu
Presence of
r weight lar
HIV proteins weight

Western blot : separation of proteins by gel

electrophoresis
 * The polyacrylamide gel
electrophoresis
The patient's Placed in an electric
serum is placed field, proteins migrate
on one edge of the as a function of their
plate SDS-PAGE molar mass

High Low
molecula molecu
Presence of
r weight lar
HIV proteins weight

Western blot : separation of proteins by gel

electrophoresis
* The polyacrylamide gel
electrophoresis

Picture of a protein separation gel SDS-PAGE system polyacrylamise

 * Transfer
Membrane
The proteins are transferred from the gel to a nitrocellulose membrane or PVDF, to make them
accessible to detection by antibodies.

The membrane is placed face-to-face with the gel, and an electric current is applied to large plates on
one side.

Charged proteins migrate from the gel to the membrane by keeping the organization on which they
had in the gel.

The nitrocellulose membranes as PVDF are 'sticky', binding proteins non-specifically (that is to say,
they bind all the proteins present in the sample in the same way)

Protein binding to the membrane occurs through hydrophobic and ionic interactions between the
membrane and proteins.
 * Transfer
blocki
Membrane
ng
The membrane having been chosen for its properties of non-specific binding, as both the target
protein that antibodies are proteins.

The site blocking non-specific interactions between the membrane and the antibody is produced by
immersing the membrane in a dilute solution of protein, mostly of the SBA in the presence of
detergent, typically Tween for one hour at room temperature.

Proteins in dilute solution bind to the membrane at all sites not occupied by the target protein. In this
way, when antibodies are applied in the next step, they can (ideally) more focus on the membrane as
the binding sites of the target protein.
 * Transfer Membrane

Nitrocellulose Immersion of the

membrane that nitrocellulose membrane
absorbs viral in a solutioncontenant
proteins separated anti-HIV proteins

Protein
s
Antibo
dy

Western blotting: Transfer to nitrocellulose and

antibody binding

*
Detectio
n
During detection, the membrane is "probed" for the protein of interest with antibodies, then linked
to an enzyme which emits a signal photometric or colorimetric, or photons. For several reasons.

This typically takes place in two stages, although one-step methods are available for certain
applications.
 *
Detectio
Two-step method
Primary
n
antibodies
A normal immune response is exploited to generate antibodies that are harvested and used as
detection tools with both high sensitivity and good specificity, binding directly to protein - hence their
name antibody "primary" .

Dilute solution of primary antibody

membrane incubated with gentle stirring

hang 30 minutes to overnight.

*
Detectio method
Two-step
Secondary
n
antibodies
Rinsing the membrane to remove unbound primary antibody.

The membrane is exposed to another antibody directed against the primary antibody.

The secondary antibody is usually linked to biotin or an enzyme that allows visual identification of the
protein of the membrane.
*
Two-step
Detectionmethod
 *
Two-step
Detectionmethod
Patient's Viral proteins
serum separated and
stained with
labeled antibodies

The 2nd series of

antibodies to the
first fine and it If the color appears, the test is
shows that the positive: the patient is HIV
enzyme is attached positive

Western blot: 2nd series of antibodies

and staining

*
One-step
Detectio method
n

These systems require a detection antibody that can both detect the protein of interest and issue a
detectable signal, which are often available for known protein markers.
 *
Colorimetric
Detectio
detection
n
This method depends on, the presence of a substrate which reacts to the trigger present on the
secondary antibody (such as peroxidase).

The substrate will be converted by the enzyme to emit a colored reaction product, visible on the
membrane.

The protein concentration is evaluated by densitometry, evaluation of the intensity of the band or
spectrophotometrically.

*
chemiluminescence
Detectio
detection
n
This method requires, during the incubation of western blot, the presence of a substrate which
emits light after exposure to trigger present on the secondary antibody.

The light emitted is used to impress a photographic film.

The image is analyzed by densitometry, which evaluates the relative rate of labeling of the protein.

The method called "enhanced chemiluminescence detection" ("enhanced chemiluminescent" - ECL) is

considered one of the most sensitive detection for the analysis of western blots.
 *
Detection
chemiluminescence
detection

*
chemiluminescence
Detectio
detection
n
 *
Detection by
Detectio
autoradiography
n

The radioactive labeling does not require enzyme substrate.

It allows the use of films used in medical imaging x-ray

This method is more or less in decline, but still sometimes used in certain circumstances.
 * Detection
Detection by autoradiography

The film is placed on the western blot

Development when exposed to the marker

Creation of dark regions

Strips of the protein of interest

 *
Detectio
Fluorescence
n
detection
Fluorescence refers to the excitation of a molecule from one stable state to an excited state, and its
return to baseline by emitting radiation in the electromagnetic spectrum of a given wavelength, and
specific of the molecule.

The probe coupled to the antibody is excited by a monochromatic beam and the resulting emission is
then detected by a photosensor, for example a CCD camera.

Conclusi
on
Step 1:
Preparation of protein samples (protein denaturation and reduction)

Step 2:
Separation of proteins according to their size by SDS PAGE electrophoresis.

Step 3:
Transfer of proteins onto a membrane (nitrocellulose).

Step 4:
Saturation of the membrane
This step helps prevent nonspecific binding of antibodies on the membrane.
 Conclusi
on
Step 5:
Detection: A solution of primary antibody was incubated with the membrane.

Step 6:
Adding the secondary antibody

Step 7 :
Revelation