Western Blotting Reagents

Western blotting (also called western blot) is a common experimental technique used to confirm the expression of specific proteins in a sample. Fujifilm Wako offers products essential for western blotting, such as the luminescent substrate of peroxidase (ImmunoStar), the reagent to improve the signal-to-noise ratio (Immuno-enhancer), and the membrane for blotting (ClearTrans).

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Principle of Western Blotting

In western blotting, protein samples are separated in a polyacrylamide gel by electrophoresis and transferred to a membrane. Specific proteins are then detected by an antigen-antibody reaction.

First, protein samples are separated according to molecular weight by SDS-PAGE. After electrophoresis, a membrane (such as nitrocellulose or polyvinylidene fluoride (PVDF)) is placed in close contact with the gel, and proteins in the gel are transferred to the membrane by applying voltage.

Next, the membrane with transferred proteins is probed with a primary antibody against the target protein. The membrane is washed, and the primary antibody is then probed with a secondary antibody labeled with horseradish peroxidase (HRP) or other enzyme and detected by chromogenic or chemiluminescence reaction of the enzyme.

Thus, using western blotting, specific proteins can be detected based on the molecular weight and antibody specificity. This is one of the most frequently performed experiments in life science research.

Method of Western Blotting

Western blotting mainly consists of four steps:

  1. Sample preparation
  2. Electrophoresis
  3. Transfer to a membrane
  4. Detection with antibodies

Each of these steps is described below.

Sample Preparation

The first step in western blotting is to prepare a cell or tissue lysate containing the target protein.

First, cells or tissues are washed with phosphate-buffered saline (PBS). Cells are lysed in a lysis buffer, and proteins are solubilized following sonication. Tissue samples are cut into small pieces with scissors and homogenized with a Teflon homogenizer or a blade homogenizer, depending on the type of tissue.

SDS-PAGE sample buffer is added to the lysate, and the sample is heat-denatured before electrophoresis.

Electrophoresis: SDS-PAGE

The purpose of SDS-PAGE is to separate proteins in the sample based on their molecular weight. Polyacrylamide gel is used for the separation.

Each protein has its own unique 3D structure and charge. If electrophoresis is performed without denaturation, proteins cannot be separated based on their molecular weight because their mobility within the gel is affected by the 3D structure. The lysate is heat-treated in an SDS-PAGE sample buffer containing sodium dodecyl sulfate (SDS) and a reducing agent. The reducing agent cleaves the disulfide bonds of proteins and disrupts the 3D structure. Subsequently, SDS binds to proteins, giving them a net negative charge.

After sample preparation is complete, the polyacrylamide gel is set in the electrophoresis apparatus, and the electrophoresis chambers are filled with the running buffer for SDS-PAGE. Depending on the current and voltage condition, electrophoresis takes approximately 1 hour.

When heat-treated samples are subjected to SDS-polyacrylamide gel electrophoresis (PAGE), negatively charged proteins migrate from cathode to anode. Proteins with smaller molecular weight have greater mobility (i.e., migrate farther) because they move through the gel more easily.

In other words, these processes allow precise separation of proteins in order of molecular weight. It is important to use the lysate immediately after the heat-treatment in the presence of a reducing agent and SDS.

Transfer

Comparison of Transfer Systems

Proteins in the gel have a negative charge due to SDS, and applying voltage causes the proteins to migrate toward the anode. In electrophoresis (SDS-PAGE), proteins migrate through the gel by applying voltage and are separated by molecular weight due to the difference in the rate of migration. In this transfer step, voltage is applied to induce the proteins to migrate from the gel to the membrane. In both cases, negatively charged proteins migrate toward the anode.

The purpose of the transfer is to induce the proteins to move out of the polyacrylamide gel to be relocated firmly on the membrane . This allows antibodies to bind to the proteins.

There are two main types of transfer methods: semi-dry and tank (wet) types.

Type Volume of Buffer Transfer Time Application
Semi-dry 30-100 mL 0.1-1h Suitable for low to moderate molecular weight proteins
Tank (wet) 300-500 mL 1.5-3h High molecular weight proteins can be analyzed. Easier to obtain high-quality results regardless of molecular weight. Less prone to uneven transfer.

In the semi-dry method, the gel is placed between two pieces of filter paper (blotting paper) soaked with transfer buffer and then placed between electrodes of the semi-dry transfer device, and electricity is applied. The transfer time is short, and only a small amount of transfer buffer is required. Note that if the transfer time is too long, the transfer buffer dries out, and the filter paper and gel could be burned by the electric current.

In the tank method, proteins in the gel are transferred to the membrane in the transfer buffer. A large amount of transfer buffer is required to completely submerge the gel. It also takes more than 1.5 hours for transfer, but the transfer efficiency is higher than that of the semi-dry method and is recommended for analyzing high molecular weight proteins.

Tips: Why is the transfer efficiency lower for high molecular weight proteins?

As mentioned in the electrophoresis section, smaller molecular weight proteins migrate faster in polyacrylamide gel , and larger proteins migrate more slowly. Similarly, proteins migrate within polyacrylamide gel during transfer, and proteins with larger molecular weight take longer to migrate out of the gel.

When the band of interest is not visible in western blotting, staining the gel after transfer with CBB or silver stain can be tried. Many proteins may remain in the gel. In such a case, the problem is often solved by increasing the transfer time or by changing the method from the semi-dry to the tank method.

Comparison of Membrane for Blotting

PVDF and nitrocellulose membranes are mainly used for western blotting.

Advantage Disadvantage
PVDF (polyvinylidene fluoride) • High physical strength and tear resistance
• High chemical resistance
• Strong adsorption to proteins
• Requires prewetting with methanol before use
Nitrocellulose • Inexpensive • Physical strength is low: easily torn
Tips: Blotting membrane suited for re-probing

When multiple antigen-antibody reactions are performed on a single membrane (re-probing), the membrane is treated with a stripping solution that removes the antibody from the antigen. If stripping is planned, PVDF membrane should be used. The stripping reagent could damage the membrane, and PVDF is better suited because of its physical strength and chemical resistance.

Transfer Buffer

The most common transfer buffer consists of Tris, glycine, and methanol. If the target protein has high molecular weight (e.g., > 100 kDa), SDS (sodium dodecyl sulfate) may be added. High molecular weight proteins move slowly in the gel and are more prone to remain in the gel than low molecular weight proteins, resulting in lower transfer efficiency. Reducing the methanol concentration to 10% or less and adding 0.1% SDS may improve the transfer efficiency.

Proteins Methanol Concentration SDS
High molecular weight proteins 10% or less Added
Low molecular weight proteins 20% or more Not added
Tips: Methanol in transfer buffer

Methanol in the transfer buffer promotes adsorption of proteins to the membrane by reducing the interaction between proteins and SDS. However, it reduces the elution of proteins from the gel, which could decrease the transfer efficiency of high molecular weight proteins.

Immunodetection: Antigen-antibody Reaction

Blocking

Blocking is performed before the addition of the primary antibody to the membrane with transferred proteins. Blocking reduces the background signal by preventing nonspecific binding of primary antibodies to the membrane.

Results vary greatly depending on the blocking solution used. Commonly used blocking solutions are 1-5% bovine serum albumin (BSA) and 1-5% skim milk2). Regarding the type of blocking agent, some antibodies react with BSA or proteins in skim milk. If that is the case, a protein-free blocking solution, consisting mainly of polymers, is used.

The concentration, blocking time, and type of blocking agent in the blocking solution should be changed depending on the antibodies used. A low concentration of blocking solution is sufficient for antibodies with low nonspecific binding. If the concentration is too high, the background signal may be reduced, but the signal of the target protein is also weakened. If the blocking time is too short, the blocking effect will be weak, and if it is too long, the signal of the target protein will also be weakened.

Generally, membranes are immersed in a blocking solution and incubated with shaking at room temperature for 30 minutes to 1 hour. After blocking, the membrane is immersed in TBS-T (0.1% Tween 20) or PBS-T (0.1% Tween 20) with shaking and washed.

Antibody Reaction

After blocking, the membrane is probed with an antibody against the target protein (primary antibody) and an enzyme-linked antibody (secondary antibody).

Antibodies are sold in high concentrations and need to be diluted before use. PBS-T or blocking solution is used for dilution. The concentration of the primary antibody is important and should be optimized empirically, while referring to the literature and the manufacturer's recommendations. If the concentration is too low, the target protein cannot be detected. If the concentration is too high, nonspecific reactions may occur and unrelated proteins may also be detected.

For overnight antibody reactions, the membrane is immersed in a diluted primary antibody solution and incubated with shaking at 4°C. If the reaction is to be completed within several hours, the membrane is incubated at room temperature.

After probing with the primary antibody, the membrane is washed with a washing solution, such as PBS-T, three times for 5 minutes each.

Detection

The band of the target protein is detected using the reaction between the enzyme linked to the secondary antibody and a chromogenic or luminescent substrate. If the primary antibody is enzyme-linked, the secondary antibody is not required.

The most common linked enzyme is horseradish peroxidase (HRP). It is also referred to simply as peroxidase (POD).

After the reaction with the primary antibody, the membrane is incubated in a diluted secondary antibody solution. The appropriate secondary antibody should be selected according to the animal (host) species of the primary antibody.

Example: If the animal (host) species of the primary antibody is mouse, a secondary antibody against mouse-derived antibodies such as Goat anti-mouse IgG-HRP should be selected.

The substrate is added to the HRP linked to the secondary antibody, and the resulting chemiluminescence is detected by a digital imager, such as a CCD camera, or by X-ray film.

Since the chemiluminescence becomes weaker with time, images should be taken as soon as possible after the reaction between the chemiluminescent substrate and the labeled secondary antibody. If no bands are visible, the exposure time should be increased. The band may become detectable if a low sensitivity is an issue.

References

  1. Nishikata, T.: “Bio Experiment Illustrated 5 ”, Shujunsha, Japan, (1996). (Japanese)
  2. Protein Experiment Notebook vol.2 4th ed.” ed. by Okada, M., Miki, H., Miyazaki, K., Yodosha, (2011). (Japanese)

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