Western Blot Method - Animated Video

Summary

Western 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

00:00

🔬 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.

05:03

🚀 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.

10:09

🌟 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.

Keywords

💡Western Blotting

Western blotting is a laboratory technique used to detect specific proteins in a sample. It involves separating proteins by size through electrophoresis, transferring them onto a membrane, and then identifying them using antibodies. The process is crucial for molecular biology research and diagnostics, as it allows for the detection and analysis of proteins of interest from complex mixtures. In the script, it is the central technique being described, with each step of the process explained in detail.

💡SDS-PAGE

SDS-PAGE, or sodium dodecyl sulfate polyacrylamide gel electrophoresis, is a method used to separate proteins based on their molecular weight. It is the first step in Western blotting where proteins are denatured and linearized to allow for uniform migration through the gel matrix. The script explains that SDS is added to the sample to give all proteins a uniform negative charge, enabling their separation during electrophoresis.

💡Protein Denaturation

Protein denaturation is the process by which proteins lose their native structure due to the disruption of non-covalent forces that maintain their shape. In the context of Western blotting, SDS denatures proteins, allowing them to be separated based on size rather than shape. The script mentions that SDS disturbs hydrogen-bonding, hydrophobic, and ionic interactions, which are responsible for the protein's three-dimensional structure.

💡Betamercaptoethanol

Betamercaptoethanol is a reducing agent used in Western blotting to cleave disulfide bonds within proteins. Disulfide bonds are covalent linkages that help stabilize the protein's structure. By breaking these bonds, betamercaptoethanol contributes to the denaturation process, ensuring that proteins are linearized for separation on the gel. The script specifies its role in the sample preparation step.

💡Polyacrylamide Gel

A polyacrylamide gel is a gel matrix used in electrophoresis to separate proteins based on their size. It is formed by polymerization between two glass plates and serves as the medium through which proteins migrate during SDS-PAGE. The script describes the gel as being produced by polymerization and used to separate proteins in a size-dependent manner.

💡Electroblotting

Electroblotting is the process of transferring separated proteins from the polyacrylamide gel to a membrane, such as nitrocellulose or polyvinylidene difluoride. This step is crucial for the detection of the target protein using antibodies. The script details the transfer process, which involves equilibrating the gel in transfer buffer and using an electric current to pull the proteins onto the membrane.

💡Blocking Solution

A blocking solution is used to prevent non-specific binding of antibodies to the membrane during the detection phase of Western blotting. It typically contains proteins like bovine serum albumin (BSA) that occupy the membrane surface, leaving only the target protein available for antibody binding. The script mentions the use of a blocking solution containing BSA to prepare the membrane for antibody incubation.

💡Primary Antibody

The primary antibody is a specific antibody that binds to the target protein of interest on the membrane. It is the first antibody used in the detection phase of Western blotting and is crucial for identifying the protein. The script describes the incubation of the membrane with the primary antibody, which then binds specifically to the target protein.

💡Secondary Antibody

The secondary antibody is used to bind to the primary antibody that is already attached to the target protein. It often has a reporter enzyme, such as horseradish peroxidase, attached to it, which allows for the detection of the primary antibody and, by extension, the target protein. The script explains that the secondary antibody recognizes and binds to the primary antibody, facilitating the detection process.

💡Chemiluminescent Detection

Chemiluminescent detection is a method used to visualize the presence of the target protein on the membrane. It involves the use of a substrate that reacts with a reporter enzyme, such as horseradish peroxidase, to produce light. The intensity of the light is proportional to the amount of the target protein, allowing for quantification. The script describes the use of a chemiluminescent substrate, such as luminol, to detect the target protein after the secondary antibody binding.

💡Densitometry

Densitometry is the process of measuring the optical density of bands on a Western blot, which allows for the quantification of the target protein. It involves capturing a digital image of the blot using a CCD camera and analyzing the intensity of the light signals from the bands. The script mentions that after chemiluminescent detection, the membrane is analyzed by densitometry to determine the size and presence of the protein of interest.

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