Western Blot Method - Animated Video
Summary
TLDRWestern blotting is a vital cell and molecular biology technique for protein separation and detection. It involves sample preparation with a loading buffer, heating, and separation using SDS-PAGE. Proteins are denatured and uniformly charged with SDS, allowing size-based separation in a polyacrylamide gel. After electrophoresis, proteins are transferred to a membrane and detected using specific antibodies. Chemiluminescent detection with horseradish peroxidase-linked secondary antibodies and luminol substrates produces light signals proportional to the target protein's presence, enabling its identification and analysis.
Takeaways
- 🧬 Western blotting is a vital technique in cell and molecular biology for separating and detecting specific proteins from complex mixtures.
- 🔬 The process involves three main steps: protein separation by size, transfer to a solid support, and detection using antibodies.
- 🌡️ Sample preparation includes adding a loading buffer and heating to 95 degrees Celsius to denature proteins and give them a uniform negative charge.
- 🔄 SDS-PAGE is used for protein separation, where SDS (an anionic surfactant) binds to proteins and allows them to be separated by molecular weight.
- 🧲 The gel electrophoresis setup includes a gel cassette with a polyacrylamide gel, placed between two electrodes with a running buffer for current conduction.
- 📏 A molecular-weight size marker is loaded alongside samples to estimate protein sizes as they migrate through the gel.
- 🚫 Bromophenol blue, a dye in the samples, helps visualize their migration and stop the electrophoresis at the right time.
- 💧 Wet transfer is used to move separated proteins from the gel to a membrane, forming a blotting sandwich and applying an electric field for electroblotting.
- 🔍 After transfer, the membrane is blocked with bovine serum albumin (BSA) to prevent non-specific interactions with antibodies.
- 🔗 The detection of the target protein is achieved using primary and secondary antibodies, with the latter often linked to a reporter enzyme for signal amplification.
- ✨ Chemiluminescent detection is a common method, where horseradish peroxidase catalyzes luminol oxidation, producing light proportional to the target protein's presence.
Q & A
What is the primary purpose of Western blotting in cell and molecular biology?
-Western blotting is used for protein separation and detection, allowing the separation and identification of a specific protein of interest from a complex mixture of proteins, such as a cell lysate.
What are the three main steps involved in a typical Western blot technique?
-The three main steps are separation by size using SDS-PAGE, transfer of protein to a solid support such as a membrane, and detecting the target protein using antibodies.
Why is a loading buffer added to protein samples in the sample preparation step of SDS-PAGE?
-The loading buffer is added to give all proteins a uniform negative charge, as it contains SDS, betamercaptoethanol, bromophenol blue, and glycerol, which helps in the uniform migration of proteins during electrophoresis.
What role does SDS play in the denaturation of proteins?
-SDS is an anionic surfactant that denatures native proteins by disturbing non-covalent forces such as hydrogen bonding, hydrophobic interactions, and ionic interactions, and it binds uniformly to proteins, making their intrinsic charges negligible compared to the negative charges from SDS.
Why is betamercaptoethanol used in the loading buffer?
-Betamercaptoethanol is a reducing agent used to cleave disulfide bonds in proteins, which is necessary for complete denaturation and proper separation during electrophoresis.
How does the polyacrylamide gel in SDS-PAGE work for protein separation?
-The polyacrylamide gel allows proteins to be separated based on their molecular weight. Smaller proteins migrate more easily through the gel mesh, while larger proteins are retained and migrate more slowly.
What is the purpose of bromophenol blue in the loading buffer?
-Bromophenol blue is a dye that helps visualize the sample in the well and serves as a tracking dye to monitor the progress of the electrophoresis.
How is the transfer of proteins from the gel to a membrane performed?
-The transfer is performed using a wet transfer method, where the gel is equilibrated in transfer buffer and then assembled into a blotting sandwich with the membrane and other components before being subjected to electroblotting.
What is the function of the blocking solution used after the protein transfer to the membrane?
-The blocking solution, often containing bovine serum albumin (BSA), is used to prevent non-specific interactions between the membrane and the antibody used for detecting the target protein.
How does the detection of the target protein using antibodies work in Western blotting?
-The detection involves incubating the membrane with a primary antibody specific to the target protein, followed by a secondary antibody that recognizes and binds to the primary antibody. The secondary antibody is often linked to a reporter enzyme for visualization, such as horseradish peroxidase in chemiluminescent detection.
What is the final step in detecting the target protein on the Western blot?
-The final step is incubating the membrane in a solution containing the substrate for the reporter enzyme, which produces a luminescent signal proportional to the amount of target protein present. The signal is then captured by a CCD camera for analysis.
Outlines
🔬 Western Blotting Technique Overview
The first paragraph introduces the Western blotting technique, a vital method in cell and molecular biology for protein separation and detection. It involves separating a specific protein from a mixture, such as a cell lysate, through steps including size separation via SDS-PAGE, protein transfer to a solid support, and antibody-based detection. The process begins with sample preparation, where proteins are given a uniform negative charge using a loading buffer containing SDS and other components. This is followed by heating to denature proteins and disrupt their native conformations. SDS-PAGE uses a polyacrylamide gel within a cassette, with proteins migrating through the gel towards the positive electrode based on their molecular weight. The use of a molecular-weight size marker helps estimate protein sizes during electrophoresis.
🚀 Detailed Process of Protein Transfer in Western Blotting
The second paragraph delves into the detailed steps of transferring separated proteins from the gel to a membrane, a critical phase in Western blotting known as electroblotting. It starts with equilibrating the gel in transfer buffer and setting up a blotting sandwich with a gel holder cassette, fiber pads, filter paper, and a nitrocellulose or PVDF membrane. The proteins are then transferred onto the membrane using an electric current, ensuring they retain their organization from the gel. After transfer, the membrane is blocked with a solution containing bovine serum albumin (BSA) to prevent non-specific interactions before being incubated with primary antibodies specific to the target protein. Following this, the membrane is washed to remove unbound primary antibodies and then incubated with a secondary antibody that recognizes the primary antibody.
🌟 Detection and Analysis of Proteins in Western Blotting
The third paragraph focuses on the detection and analysis of proteins in Western blotting. It describes the use of chemiluminescent detection, where the secondary antibody is linked to a reporter enzyme like horseradish peroxidase (HRP). HRP catalyzes the oxidation of luminol in the presence of hydrogen peroxide, resulting in light emission proportional to the amount of HRP-conjugated secondary antibody, thus indicating the presence of the target protein. The membrane is then analyzed using densitometry, where a CCD camera captures a digital image of the blot, allowing for the determination of the protein's size and presence in the samples. This method provides a quantitative measure of protein expression levels.
Mindmap
Keywords
💡Western Blotting
💡SDS-PAGE
💡Protein Denaturation
💡Betamercaptoethanol
💡Polyacrylamide Gel
💡Electroblotting
💡Blocking Solution
💡Primary Antibody
💡Secondary Antibody
💡Chemiluminescent Detection
💡Densitometry
Highlights
Western blotting is a crucial technique in cell and molecular biology for protein separation and detection.
It allows for the separation and identification of a specific protein from a complex mixture, such as a cell lysate.
The process involves separation by size, transfer to a solid support, and detection using antibodies.
SDS-PAGE is utilized for separating macromolecules in a sample based on size.
Sample preparation involves adding a loading buffer containing SDS and other components to protein samples.
SDS provides a uniform negative charge to proteins, facilitating their separation by molecular weight.
Heating samples to 95 degrees Celsius is necessary for protein denaturation.
Proteins are large biomolecules formed by amino acids linked by peptide bonds and folding into specific conformations.
SDS disrupts non-covalent forces in proteins, such as hydrogen bonds and hydrophobic interactions.
Betamercaptoethanol is used to cleave disulfide bonds in proteins.
Gel electrophoresis separates proteins based on molecular weight in a polyacrylamide gel.
The gel is prepared between two glass plates and subjected to an electric field for protein migration.
A molecular-weight size marker is used alongside samples to estimate protein sizes.
Bromophenol blue and glycerol in the samples aid in visualization and sample application into the gel wells.
Proteins migrate towards the positive electrode, with smaller proteins moving faster through the gel matrix.
After separation, proteins are transferred from the gel to a membrane using wet transfer or electroblotting.
The transfer process involves creating a blotting sandwich and using an electric current to move proteins onto the membrane.
Detection of the target protein is achieved using antibodies and a blocking solution to prevent non-specific interactions.
Primary and secondary antibodies are used sequentially to bind specifically to the target protein and enable detection.
Chemiluminescent detection with horseradish peroxidase and luminol is a common method for visualizing proteins on Western blots.
The luminescence produced is proportional to the target protein's presence, allowing for its detection and analysis.
Densitometry and a CCD camera are used to capture a digital image of the Western blot for further analysis.
Transcripts
Western blotting is an important technique used in cell and molecular biology
for protein separation and detection
it enables the separation and identification of a specific protein of interest
from a complex mixture of proteins
for example a cell lysate
A typical Western blot technique includes the following steps
separation by size, transfer of protein to a solid support
and detecting target protein using antibodies
to separate the macromolecules in a sample
SDS-PAGE technique can be used
The first step in the SDS-Page procedure is sample preparation
So that, a loading buffer containing SDS, betamercaptoethanol
bromophenol blue and glycerol is added to the protein samples
the loading buffer is used in order to give all proteins present a uniform negative charge
since proteins can be positively, negatively, or neutrally charged
Once the loading buffer is added
the samples are then heated to 95 degrees Celsius
Proteins are large biomolecules, consisting of one or more long chains of amino acid residues
they are formed by linking amino acids with peptide bonds
and fold into specific spatial conformations driven by a number of interactions
such as Hydrogen bonds, Hydrophobic interaction, Disulfide bonds, Ionic bonds.
SDS is an anionic surfactant that contain a polar head group with a net negative charge
at the end of a long hydrophobic carbon chain
SDS denatures the native proteins by disturbing the non-covalent forces
include hydrogen-bonding, hydrophobic and ionic interactions
While the reducing agent betamercaptoethanol is used to cleave the disulfide bonds
Also, SDS binds fairly uniformly to the protein
and the intrinsic charges of this protein become negligible
when compared to the negative charges contributed by SDS
this treatment brings the folded proteins down to linear molecules, with net negative charge
therefore, these proteins can be separated in a polyacrylamide gel based on their molecular weight
The gel is produced by polymerisation between two glass plates anchored vertically in a cassette
The gel cassette is placed vertically between two electrodes
positive electrode located at the bottom of the gel
whereas negative electrode is positioned at the top of the gel
Next, the gel is inserted into a chamber
then a running buffer is poured to allow the conduction of current through the gel
SDS is also present in the gel and in the running buffer, to make sure that
once the proteins are denatured, they stay that way throughout the run
During separation, a molecular-weight size marker is usually loaded onto the gel
This consists of proteins of known sizes
and thereby allows the estimation of the sizes of the proteins in the actual samples
which migrate in parallel in different tracks of the gel
Each sample is added into its own well in the gel
as we have seen previously, bromophenol blue and glycerol are present in the samples
bromophenol blue is a dye, that is useful for visualizing the sample in the well
while, Glycerol increases the density of a sample and it is used to fall the sample into the well
after the sample application procedure, an electric field is applied across the gel
causing the negatively charged proteins to migrate across the gel
away from the negative electrode, and towards the positive electrode
Also, Small proteins migrate relatively easily through the mesh of the gel
while larger proteins are more likely to be retained and thereby migrate more slowly
Due to the relatively small molecule size of bromophenol blue, it migrates faster than proteins
and by optical control of the migrating-colored band
the electrophoresis can be stopped before the samples have completely migrated through the gel and leave it
Once the proteins are separated, the gel cassette is removed from the electrophoresis tank
Next, the glass plates are removed
and, the top portion containing the wells is cut off from the gel
In the next step, the separated proteins will be transferred
from inside the gel to an appropriate membrane
When performing a wet transfer, the gel is first equilibrated in transfer buffer
so that, it is placed in a tray with the blotting buffer, on a rocking platform
To make a blotting sandwich, a gel holder cassette is immersed in the transfer buffer
with the black side down and the white side up, and out of the buffer
Next, a fiber foam pad is immersed in the transfer buffer
Then, it is laid on the black side of the gel holder cassette
After that, a piece of a filter paper is soaked in the transfer buffer
Then, it is placed on top of the fiber pad
once the gel has been equilibrated in the transfer buffer
it is carefully placed on the blotting paper
For the transfer, a nitrocellulose membrane or a polyvinylidene difluoride membrane can be used
the membrane is soaked in the transfer buffer
then, it is placed squarely on the gel
next, a second sheet of blotting paper is wetted with the transfer buffer
Then, it is placed on top of the membrane
Finally, a second fiber pad is immersed in the transfer buffer
Then, it is laid on top of the blotting paper
Next, the gel holder cassette is closed and locked with a clamp
The most commonly used method for transferring the proteins is called electroblotting
During this transfer, an electrode assembly with positive electrode and negative electrode is used
The gel holder cassette is inserted into the module
ensuring that the cassette is properly positioned from negative to positive
The inner module is placed into the electrophoresis chamber
then, the chamber is filled with the blotting buffer
Next, a lid is placed on the electrophoresis tank
and electrical leads are connected to the power supply to run the blot
Electroblotting uses an electric current to pull the negatively charged proteins from the gel
towards the positively charged electrode, and into the membrane
The proteins move from within the gel onto the membrane
while maintaining the organization they had within the gel
when the run is complete, the electrophoresis chamber is disassembled
and the inner module is removed
Then, the gel holder cassette is removed and opened
Next, starting with the fiber pad, each layer is removed until reaching the membrane
After the electrotransfer of the proteins on the membrane
the next step is the detection of our protein of interest using antibodies
first of all, the membrane is soaked in a blocking solution
which contains bovine serum albumin
this step must be taken to prevent the interactions between the membrane
and the antibody used for detection of the target protein
once the blocking solution has been added, the membrane is incubated on a rocking platform.
The BSA protein attaches to the membrane in all places where the target proteins have not attached
Thus, when the antibody is added, it cannot bind to the membrane
and therefore the only available binding site is the specific target protein
After the incubation is complete, the blocking solution is removed
then a solution containing the primary antibody is poured into the tray
next the membrane is incubated under gentle agitation
The primary antibody binds to the specific target protein
Following incubation, the primary antibody solution is removed
then a wash buffer is poured
next the membrane is incubated on the rocking platform
the membrane is washed several times in wash buffer to remove unbound primary antibody
and thereby minimize background
after washing the membrane, the wash buffer is removed
then, a solution containing the secondary antibody is added into the tank
next the membrane is incubated under gentle agitation
The secondary antibody recognises and binds to a specific portion of the primary antibody
Following incubation, the secondary antibody solution is removed
then, the wash buffer is poured into the tank
next the membrane is incubated on the rocking platform
the membrane is washed several times in wash buffer to remove unbound secondary antibodies
It is crucial to thoroughly wash the membrane at this step
After rinsing the membrane to remove unbound secondary antibody
the membrane is incubated in a solution containing the substrate.
Chemiluminescent western blot detection is the most frequently used method
to detect proteins on Western blots
To allow detection of the target protein
the secondary antibody is commonly linked to a reporter enzyme such as horseradish peroxidase
The most popular Western blot substrates are luminol-based
in the presence of a hydrogen peroxide, Horseradish peroxidase enzyme catalyses the oxidation of luminol
to excited state product called 3-aminophthalate
This product decays to a lower energy state by releasing photons of light at 425 nm
the production of luminescence is proportional to the amount of horseradish peroxidase-conjugated secondary antibody
and therefore, indirectly measures the presence of the target protein
After incubation, the membrane is placed in a clear plastic wrap to prevent drying
The membrane is analysed by densitometry
where the resulting light signal is detected by CCD camera which captures a digital image of the western blot
according to the obtained bands, we can determine the approximate size of our protein of interest
as well as its presence or absence in each sample
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