Western blot is a commonly used method for protein analysis. It can be used for qualitative and semi-quantitative protein analysis. For the accomplishment of the western blot, there are three elements, separation of proteins by size, transferring proteins to a solid support, and marking proteins by primary and secondary antibodies for visualization.
2. Western blot was introduced in 1979,
which is a commonly used method for
protein analysis.
It can be used for qualitative and semi-
quantitative protein analysis.
THREE ELEMENTS
• Separation of proteins by size
• Transferring proteins to a solid support
• Marking proteins by primary and secondary antibodies
for visualization
Western Blot
3. Principles
SDS-PAGE allows protein samples to be separated and transferred to a solid
support. The solid support can absorb the protein and keep its biological
activity unchanged.
Only the proteins to be studied can specifically bind to the primary antibody
to form an antigen-antibody complex.
The labeled secondary antibody that binds to the primary antibody forms
an antibody complex that can indicate the location of the primary antibody,
both the location of the protein being studied.
5. Sample preparation
Cells Tissues
Lysate/Extracts
Proteins can be extracted from different samples, such
as tissues or cells.
Tissues are first broken down by the mechanical
invention, such as homogenizer or sonication.
Protease and phosphatase inhibitors are commonly used
to prevent the digestion of the sample at cold
temperatures.
Detect the concentration of proteins: a
spectrophotometer .
6. Gel electrophoresis
-
+
-
+
High Molecular weight
Low Molecular weight
Stacking gel
Separating gel
The most commonly used gel is polyacrylamide gels (PAG) and buffers loaded with sodium dodecyl
sulfate (SDS).
The smaller the known weight of proteins is, the higher percentage of gels should be used.
5 to 2,000 kDa
7. Proteins transfer
Sponge
Filter Papers
Blotting Membrane
Gel
Filter Papers
Sponge
+
Electroblotting, which uses an electric field oriented perpendicular
to the surface of the gel, to pull proteins out of the gel and move
into the membrane.
The membrane is placed between the gel surface and filter.
The transfer sandwich is created as follows: a sponge, filter
papers, the gel, a membrane, filter papers, a sponge.
Proteins are moved from within the gel onto a solid support
membrane made of nitrocellulose (NC) or polyvinylidene
difluoride (PVDF) to make the proteins accessible to antibody
detection.
Wet conditions are usually more reliable as it is less likely dry out
the gel.
8. Blocking
Prevent antibodies from binding to the membrane
nonspecifically.
The most commonly used typical blockers are
BSA and nonfat dry milk.
When the membrane is placed in the dilute
solution of proteins, the proteins attach to all
places in the membrane where the target proteins
have not attached.
The “noise” in the final product of the western blot
can be reduced and result in clearer results.
9. Antibody incubation
Membrane Membrane
Primary antibody
Membrane
Labeled
secondary antibody
The primary antibody binds to target proteins when the primary antibody is incubated with
the membrane.
The choice of a primary antibody depends on the antigen to be detected.
Washing the membrane with the antibody-buffer solution is helpful for minimizing
background and removes unbound antibodies.
After rinsing the membrane, the membrane is exposed to the specific enzyme-conjugated
secondary antibody.
When performing secondary antibody incubation, the labeled secondary antibody can bind
to the primary antibody which has reacted with target proteins.
10. Protein detection and visualization
Colorimetric
detection
Chemiluminescent
detection
Radioactive
detection
Fluorescent
detection
A substrate reacts with the enzyme that is bound to the secondary antibody to
generate colored substance. It enables us to know the densitometry and location of the
targets protein.
And the size approximations are taken by comparing the proteins bands to the marker.
Electrochemiluminescence
(ECL) system
11. Option 03
Western Blot & Electrical
Transfer
Option 04
2D Blue Native for Complex
Analysis
Option 01
2D Electrophoresis
Option 02
SDS-PAGE, IEF and native
PAGE analysis
Protein Gel
and
Imaging
Analysis
Our services
At Creative Proteomics, we can provide an integrated solution for the identification of low abundance proteins in
complex biological samples.
Western blot was introduced in 1979, which is a commonly used method for protein analysis. It can be used for qualitative and semi-quantitative protein analysis. For the accomplishment of the western blotting, there are three elements, separation of proteins by size, transferring proteins to a solid support, and marking proteins by primary and secondary antibodies for visualization.
Western blot is performed using polypropylene gel electrophoresis. In this method, proteins can be detected, the "probe" is an antibody, and the secondary antibody is used for coloration. SDS-PAGE allows protein samples to be separated and transferred to a solid support, such as nitrocellulose (NC) or polyvinylidene difluoride (PVDF) membrane. The solid support can absorb the protein and keep its biological activity unchanged. The transferred solid support membrane is called a blot and is treated with a protein solution to block the hydrophobic binding site on the membrane. The membrane is treated with the antibody (primary antibody) of the target proteins. Only the proteins to be studied can specifically bind to the primary antibody to form an antigen-antibody complex. After the primary antibody is washed and removed, only the position of the target protein binds to the primary antibody. The primary antibody-treated membranes are treated with a labeled secondary antibody after washing. After treatment, the labeled secondary antibody that binds to the primary antibody forms an antibody complex that can indicate the location of the primary antibody, both the location of the protein being studied.
There are six steps involved in western blot, including sample preparation, gel electrophoresis, proteins transfer, blocking, antibody incubation, and proteins detection and visualization.
Proteins can be extracted from different samples, such as tissues or cells. Since tissue samples display a higher degree of structure, the tissues are first broken down by the mechanical invention, such as homogenizer or sonication. Protease and phosphatase inhibitors are commonly used to prevent the digestion of the sample at cold temperatures. After protein extraction, it is important to detect the concentration of proteins, which permits the mass of proteins loaded into each well. And a spectrophotometer is often used for proteins concentration.
Speaking of the gel electrophoresis,the most commonly used gel is polyacrylamide gels and buffers loaded with sodium dodecyl sulfate. Western blot uses two types of agarose gel: stacking gel that is used for concentrate all proteins in one band, and separating gel, that allows to separating proteins according to their molecular weight. Smaller proteins migrate faster in SDS-PAGE (SDS polyacrylamide gel electrophoresis) when a voltage is applied. PAGE can separate proteins ranging from 5 to 2,000 kDa according to the uniform pore size which is controlled by the Different concentration of PAG. When we choose the appropriate percentage of the separating gel, we should consider the size of the target proteins. The smaller the known weight of proteins is, the higher percentage of gels should be used.
After separating proteins by gel electrophoresis, proteins are moved from within the gel onto a solid support membrane made of nitrocellulose (NC) or polyvinylidene difluoride (PVDF) to make the proteins accessible to antibody detection. The main method for transferring proteins is called electroblotting, which uses an electric field oriented perpendicular to the surface of the gel, to pull proteins out of the gel and move into the membrane. It can be done semi-dry or wet conditions, while wet conditions are usually more reliable as it is less likely dry out the gel. As shown in the left figure, the membrane is placed between the gel surface and filter. The transfer sandwich is created as follows: a fiber pad (sponge), filter papers, the gel, a membrane, filter papers, a fiber pad.
Blocking is an important step in the Western Blot to prevent antibodies from binding to the membrane nonspecifically. The most commonly used typical blockers are BSA and nonfat dry milk. When the membrane is placed in the dilute solution of proteins, the proteins attach to all places in the membrane where the target proteins have not attached. In this way, the “noise” in the final product of the western blot can be reduced and result in clearer results.
After blocking, the primary antibody binds to target protein on the membrane when the primary antibody is incubated with the membrane. The choice of a primary antibody depends on the antigen to be detected. Washing the membrane with the antibody-buffer solution is helpful for minimizing background and removes unbound antibodies. After rinsing the membrane, the membrane is exposed to specific enzyme conjugated the secondary antibody. When performing secondary antibody incubation, the label secondary antibody can bind to the primary antibody which has reacted with target proteins. Based on the species of the primary antibody, we can choose the appropriate secondary antibody.
A substrate reacts with the enzyme that is bound to the secondary antibody to generate colored substance to enable to know the densitometry and location of the targets protein. And the size approximations are taken by comparing the proteins bands to the marker. There are several detection systems are available for protein visualization, such as colorimetric detection, chemiluminescent detection, radioactive detection, and fluorescent detection .The electrochemiluminescence (ECL) system is the most common detection methods.
The western blot is commonly used for qualitative detection of proteins and post-translational modifications (e.g. phosphorylation). In addition, it also can be used in medical diagnostics, such as the HIV-test or BSE-test.
At Creative Proteomics, we can provide an integrated solution for the identification of low abundance proteins in complex biological samples. We can provide two dimensional Electrophoresis, SDS-PAGE, IEF and native PAGE analysis, Western Blot and Electrical Transfer service, 2D Blue Native / SDS-PAGE for Complex Analysis
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