Eastern Blot- Definition, Principle, Steps, Results, Applications
Eastern blot is a biochemical technique that is used to analyze protein post-translational modifications (PTM) such as lipids, phosphates, and glycoconjugates . It is most often used to detect carbohydrate epitopes on proteins and lipids . Thus, eastern blot can be considered an extension of the biochemical technique of western blot, which is more popular and widely used .
Eastern blot is an immunoblotting technique that depends on the specificity between the protein of interest and a probe in order to identify the biomolecule of interest in a mixture of different molecules. The proteins are electrophoresed on polyacrylamide gel in order to separate them from the mixture. The proteins are then transferred to a nitrocellulose or nylon membrane, where the target molecules are detected by their specific interaction with the probes. The identification of the interaction can be made either through the use of a radioactive probe or by the use of a secondary tagged molecule like in the case of ELISA.
Eastern blotting, like all other blotting techniques, is based on the antigen-antibody interactions where the specificity and extent of interaction determine the result. Eastern blotting is similar to lectin blotting as both of them are primarily used for the detection of carbohydrate epitopes on proteins and lipids. The use of eastern blotting for the detection of smaller molecular compounds is essential as the detection of these molecules cannot be achieved by immunostaining.
Eastern blot has been slightly modified to form a new form of blotting technique called far-eastern blotting, which is used to study lipids that are separated by chromatography. Far-eastern blotting uses antibodies or lectins to stain lipids transferred to polyvinylidene fluoride membranes.
Eastern blot has many applications in the analysis of post-translational modifications in proteins and the detection of different plant products. It also helps to study the nature of interactions between different molecules by the use of ligands. Eastern blot has been extensively used to compare modifications in proteins obtained from different bacterial species.
However, eastern blot also has some limitations such as its complexity, requirement of large amount of sample, difficulty in quantification, and potential destruction of protein structure.
In this article, we will discuss the principle, steps, results, applications, and limitations of eastern blot in detail.
Explanation of the principle of Eastern Blot
Eastern blotting is a molecular biology technique that allows the detection and identification of post-translational modifications in proteins, such as glycosylation, phosphorylation, acetylation, etc. These modifications can affect the structure, function, and interactions of proteins, and are often involved in various biological processes and diseases.
The principle of eastern blotting is based on the specific binding of a probe to the modified protein of interest. The probe can be a monoclonal antibody (MAb), a lectin, a ligand, or any other molecule that recognizes the modification. The probe can also be labeled with a radioactive or enzymatic tag for detection.
The general steps of eastern blotting are as follows:
- The proteins are separated by thin layer chromatography (TLC) on a silica gel plate. TLC is a method that separates molecules based on their polarity and size. The proteins are applied to the plate and then eluted with a solvent. The solvent moves along the plate by capillary action and carries the proteins with it. The proteins with different polarity and size will migrate at different rates and form spots on the plate.
- The proteins are transferred from the TLC plate to a membrane, such as polyvinylidene difluoride (PVDF) or nitrocellulose. This can be done by pressing or heating the plate and the membrane together, or by applying an electric current through them. The transfer ensures that the proteins retain their position and orientation on the membrane.
- The membrane is treated with sodium periodate (NaIO4), which oxidizes the carbohydrate groups on the proteins and forms aldehyde groups. These aldehyde groups can react with bovine serum albumin (BSA) in alkaline conditions and form hapten-BSA conjugates on the membrane. These conjugates serve as antigens for the MAbs that recognize the modification.
- The membrane is blocked with a solution of skim milk or another protein to prevent nonspecific binding of the probe. The membrane is then incubated with the probe that binds to the modified protein of interest. The probe can be either directly labeled with a tag or indirectly detected by a secondary antibody that binds to the probe and carries a tag.
- The membrane is washed to remove any unbound probe or antibody. The tag on the probe or antibody is then visualized by adding a substrate that produces a color or radioactivity. The intensity and location of the signal indicate the presence and amount of the modified protein on the membrane.
Eastern blotting is similar to other blotting techniques, such as western blotting, northern blotting, and southern blotting, in that they all use electrophoresis, transfer, and hybridization steps. However, eastern blotting differs from them in several aspects:
- Eastern blotting uses TLC instead of gel electrophoresis to separate proteins. TLC has some advantages over gel electrophoresis, such as higher resolution, faster separation, lower cost, and less sample consumption.
- Eastern blotting detects post-translational modifications in proteins rather than their primary structure or nucleic acid sequence. Therefore, eastern blotting requires probes that are specific for the modification rather than for the protein itself.
- Eastern blotting can detect modifications in both proteins and lipids, whereas other blotting techniques are mainly used for nucleic acids or proteins.
Eastern blotting is a useful technique for studying post-translational modifications in proteins and their roles in various biological phenomena. However, it also has some limitations, such as complexity, sensitivity, specificity, and quantification issues. These will be discussed in more detail later in this article.
The following are the requirements for eastern blotting:
- Thin Layer Chromatography (TLC) Plate: This is the plate on which the sample mixture is separated by chromatography. The TLC plate can be made of silica gel, alumina, or cellulose. The choice of the plate depends on the polarity and solubility of the sample components.
- Transfer Membrane: This is the membrane on which the separated components are transferred from the TLC plate. The transfer membrane can be made of nitrocellulose, polyvinylidene difluoride (PVDF), or nylon. The choice of the membrane depends on the size and charge of the sample components.
- Sodium Periodate (NaIO4) Solution: This is the solution that is used to fix the components on the transfer membrane. Sodium periodate oxidizes the hydroxyl groups of carbohydrates and alcohols to form aldehydes, which can then react with amino groups of proteins to form covalent bonds. This ensures that the components do not diffuse or wash off from the membrane.
- Blotting Solution: This is the solution that is used to wet the transfer membrane and facilitate the transfer of components from the TLC plate. The blotting solution can be water, methanol, ethanol, or a buffer solution. The choice of the blotting solution depends on the solubility and stability of the sample components.
- Sodium Carbonate (Na2CO3)/ Sodium Bicarbonate (NaHCO3) Buffer: This is the buffer solution that is used to prepare hapten-BSA conjugate on the transfer membrane. The buffer solution maintains a pH of around 9.5, which favors the formation of Schiff base between aldehydes and amino groups.
- Bovine Serum Albumin (BSA): This is a protein that is used to conjugate with haptens on the transfer membrane. BSA has many amino groups that can react with aldehydes to form covalent bonds. BSA also serves as a blocking agent to prevent nonspecific binding of antibodies to the membrane.
- Monoclonal Antibody (MAb): This is an antibody that is specific for a particular hapten on the transfer membrane. MAb can be obtained from hybridoma cells or recombinant DNA technology. MAb can be labeled with a radioactive or enzymatic tag for detection purposes.
- Secondary Antibody: This is an antibody that binds to the primary antibody (MAb) on the transfer membrane. Secondary antibody can be obtained from a different animal species than MAb. Secondary antibody can also be labeled with a radioactive or enzymatic tag for detection purposes.
- Substrate: This is a molecule that reacts with the enzymatic tag on the secondary antibody to produce a color or luminescence signal. The choice of substrate depends on the type of enzyme used as a tag. For example, if peroxidase is used as a tag, then 3,3`,5,5`-tetramethylbenzidine (TMB) can be used as a substrate.
The following is the protocol for eastern blotting:
- The TLC plate developed after chromatography is covered with the PVDF membrane in order to transfer the contents from the plate to the membrane either by pressing or heating. The transfer of components from the TLC plate to the membrane can also be obtained by passing electric current through the structure.
- The membrane is then treated with sodium periodide to fix the components on the membrane. It is incubated in the solution for 1 hour and is washed with water.
- The membrane is immersed in BSA solution at alkaline condition (Na2CO3/NaHCO3 buffer) to prepare hapten-BSA conjugate on the membrane. It is blocked with 5% skim milk/PBS for 3 hours and is washed with PBS. The membrane was treated by MAb, which detects the hapten.
- The interaction between the MAb and the hapten forms the basis of eastern blotting for its detection. The MAb can then be detected by the addition of a second antibody labeled with peroxidase.
- Finally, a substrate is added to the membrane, which helps in the visualization of the second antibody via staining.
The procedure of eastern blotting can be summarized in the following diagram:
graph LR A(TLC plate) --> B(PVDF membrane) B --> C(NaIO4 solution) C --> D(BSA solution) D --> E(MAb) E --> F(Second antibody) F --> G(Substrate)
The result of eastern blotting depends on the type of label used in the second antibody. The positive result is indicated by the appearance of color or radioactivity. The negative result is indicated by the lack of color or radioactivity.
The color or radioactivity can be measured by different methods, such as densitometry, autoradiography, chemiluminescence, or fluorescence. These methods allow the visualization and quantification of the signal intensity, which reflects the amount and specificity of the probe binding to the target molecule.
The interpretation of the results also depends on the quality and accuracy of the experimental steps, such as the separation of the molecules on TLC plate, the transfer to the membrane, the fixation and blocking of the membrane, and the incubation with the probes and antibodies.
Some factors that can affect the results are:
- The resolution and sensitivity of the TLC plate and the membrane
- The purity and specificity of the probes and antibodies
- The concentration and incubation time of the probes and antibodies
- The background noise and interference from other molecules
- The stability and activity of the label and the substrate
To ensure reliable and reproducible results, it is important to optimize these factors and use appropriate controls and standards in each experiment.
Some examples of controls and standards are:
- Positive control: a sample that contains a known amount of the target molecule
- Negative control: a sample that does not contain the target molecule
- Blank control: a sample that does not contain any molecule
- Internal standard: a molecule that is added to each sample to normalize the signal intensity
- External standard: a series of samples that contain known amounts of the target molecule to generate a calibration curve
By comparing the signal intensity of the samples with the controls and standards, one can determine whether the target molecule is present or absent, and estimate its quantity and purity.
The result of eastern blotting is based on the specific interaction between the probe and the target molecule on the membrane. The probe can be either a radioactive label or a secondary antibody tagged with an enzyme. The detection of the probe can be done by autoradiography or colorimetric methods.
A positive result in eastern blotting indicates that the target molecule has been successfully transferred from the TLC plate to the membrane and that it has a specific binding affinity for the probe. A positive result can be visualized by the appearance of a band or a spot on the membrane corresponding to the location of the target molecule. The intensity of the band or spot can reflect the amount of the target molecule present in the sample. A positive result can also be quantified by measuring the radioactivity or color intensity of the band or spot.
A negative result in eastern blotting indicates that either the target molecule was not transferred from the TLC plate to the membrane, or that it did not bind to the probe. A negative result can be visualized by the absence of any band or spot on the membrane. A negative result can also be due to technical errors such as insufficient transfer, poor fixation, inadequate blocking, nonspecific binding, low probe concentration, or improper detection methods.
To verify the validity of the results, some controls should be included in eastern blotting. These include:
- A positive control that contains a known amount of the target molecule to ensure that the transfer, fixation, and detection steps are working properly.
- A negative control that contains no target molecule to ensure that there is no background or nonspecific binding of the probe.
- A loading control that contains a reference protein or biomolecule that is present in all samples to ensure that equal amounts of samples are loaded and transferred.
- A molecular weight marker that contains proteins or biomolecules of known sizes to estimate the molecular weight of the target molecule.
The interpretation of the results in eastern blotting should be done with caution and in conjunction with other complementary methods. Eastern blotting is a qualitative and semi-quantitative technique that can provide information about the presence and modification of biomolecules, but it cannot provide information about their function or activity. Therefore, eastern blotting should be combined with other techniques such as mass spectrometry, enzymatic assays, or functional assays to gain a comprehensive understanding of the biomolecules of interest.
Eastern blotting is a versatile technique that has various applications in molecular biology, biochemistry, and biotechnology. Some of the main applications of eastern blotting are:
- Analysis of post-translational modifications in proteins: Eastern blotting can be used to detect and characterize different types of modifications in proteins, such as glycosylation, phosphorylation, acetylation, methylation, ubiquitination, and others. These modifications can affect the structure, function, and interactions of proteins and are involved in many biological processes and diseases. Eastern blotting can help to identify the sites and extent of modifications in proteins and to compare them among different samples or conditions.
- Identification and purification of natural products: Eastern blotting can be used to identify and purify various natural products from plants, animals, fungi, bacteria, and other sources. These natural products can have medicinal, nutritional, or industrial value and can be detected by their specific interactions with probes. For example, eastern blotting has been used to identify and purify ginsenosides from ginseng, alkaloids from opium poppy, cannabinoids from cannabis, and flavonoids from licorice.
- Detection of carbohydrate epitopes in proteins and lipids: Eastern blotting can be used to detect the presence and distribution of carbohydrate epitopes in proteins and lipids. Carbohydrate epitopes are sugar moieties that are attached to proteins or lipids and can serve as recognition sites for receptors, antibodies, lectins, or other molecules. Eastern blotting can help to study the role of carbohydrate epitopes in cell signaling, adhesion, infection, immunity, and other phenomena.
- Study of molecular interactions: Eastern blotting can be used to study the nature and specificity of molecular interactions between different biomolecules. By using different probes or ligands, eastern blotting can reveal the binding affinity, kinetics, stoichiometry, and competition of interactions. For example, eastern blotting has been used to study the interactions between lectins and glycoproteins, antibodies and antigens, hormones and receptors, drugs and targets, and enzymes and substrates.
Eastern blotting is a powerful tool that can provide valuable information about the structure, function, and dynamics of biomolecules. It can also help to discover new biomarkers, drug candidates, or therapeutic targets for various applications.
Eastern blotting is a powerful technique for the detection and analysis of post-translational modifications in proteins and other biomolecules. However, like any other technique, it has some limitations that need to be considered before using it. Some of the limitations are:
- Eastern blotting is a complex and time-consuming technique that requires multiple steps and reagents. It also needs skilled personnel and specialized equipment for its execution. The technique is prone to errors and variations that can affect the quality and reliability of the results.
- Eastern blotting requires a large amount of sample for the analysis, which can be a challenge in some cases where the sample is scarce or difficult to obtain. The sample also needs to be properly prepared and stored to avoid degradation or contamination that can interfere with the detection of the target molecules.
- Eastern blotting is not very sensitive or quantitative compared to other techniques such as mass spectrometry or ELISA. The detection of the target molecules depends on the affinity and specificity of the probes and antibodies used, which can vary depending on the source and quality of the reagents. The signal intensity also depends on the type and amount of label used, which can introduce variability and bias in the results.
- Eastern blotting can alter or damage the structure and function of the target molecules during the transfer or detection process. The use of sodium periodate, for example, can oxidize some functional groups in the molecules and affect their activity or interaction with other molecules. The use of heat or electric current can also denature or degrade some proteins and affect their conformation and stability.
- Eastern blotting is limited by the availability and specificity of the probes and antibodies that can recognize the target molecules. The technique is mainly used for the detection of carbohydrate epitopes on proteins and lipids, but it cannot detect other types of modifications such as phosphorylation, methylation, acetylation, etc. The probes and antibodies also need to be validated and optimized for each specific application and sample type.
These limitations do not mean that eastern blotting is not useful or reliable, but rather that it should be used with caution and in combination with other complementary techniques that can provide more information and validation for the results. Eastern blotting is still a valuable tool for the study of post-translational modifications in proteins and other biomolecules, especially in cases where other techniques are not feasible or available.
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