You are currently viewing 46 Difference Between Western Blot and Immunoblot

46 Difference Between Western Blot and Immunoblot

Both the Western blot and the immunoblot are scientific procedures for identifying particular proteins in a sample. They are frequently employed to evaluate protein expression, recognise post-translational changes, and measure protein levels in molecular biology and biochemistry research. Although the terms are frequently used interchangeably, their definitions can vary slightly depending on the situation and academic discipline.

Scientists first used the term “Western blot” to distinguish this method from the related Southern blot (DNA) and Northern blot (RNA) methods. Using gel electrophoresis, often sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), proteins from a sample are sorted by size in a Western blot. Once the proteins have been separated, they are subsequently deposited (or “blotted”) onto a membrane, which is commonly constructed of nitrocellulose or polyvinylidene difluoride (PVDF). The spatial layout of the proteins on the gel is preserved during the transfer process.

After the proteins have been moved to the membrane, they are examined using certain antibodies that bind to the desired target protein. These antibodies have a detection signal attached to them, either an enzyme or a fluorescent dye. Following the removal of any unbound antibodies, the reaction of the detection marker with a substrate allows the presence of the target protein to be seen. This generates a signal that can be recorded using image technology, film, or chemiluminescence.

Particularly in disciplines like immunology, the terms “immunoblot” and “Western blot” are frequently used interchangeably. A technique where proteins are separated and detected depending on their responsiveness to particular antibodies is known as an immunoblot. In this context, the phrase draws attention to the immunological character of the method and the employment of antibodies to identify certain proteins.

Both Western blotting and immunoblotting are methods that separate proteins by electrophoresis, transfer them to a membrane, and then detect them using certain antibodies. The language used depends frequently on the research area and how much emphasis is placed on the immunological component of protein detection. However, in most situations, the phrases are interchanged to refer to the same core protein analysis technique.



Western Blot




Commonly referred to as Western Blot

Can be used interchangeably with Western Blot


Historical Origin

Coined in the context of Southern and Northern Blots

Term more commonly used in recent literature


Technique Purpose

Detects and analyzes specific proteins

Detects and analyzes proteins or other biomolecules


Target Molecules

Typically used for protein detection

Can be used for various targets, including proteins


Electrophoresis Step

Preceded by SDS-PAGE (Sodium Dodecyl Sulfate-PAGE)

Can be preceded by various electrophoresis methods


Transfer Method

Involves transferring proteins to a membrane (e.g., nitrocellulose or PVDF)

Similar transfer methods as Western Blot


Membrane Types

Often uses nitrocellulose or PVDF membranes

Uses various membranes depending on the application


Blocking Step

Involves blocking nonspecific binding sites

Requires blocking to prevent nonspecific binding


Primary Antibodies

Used to detect the target protein

May be used to detect specific biomolecules


Secondary Antibodies

Typically conjugated to enzymes or fluorophores

Similar use of secondary antibodies


Detection Methods

Enzymatic or chemiluminescent detection

May use various detection methods, including enzymes, fluorescence, or chemiluminescence



Can be highly sensitive for protein detection

Sensitivity can vary depending on the method used



Mainly used for protein analysis and quantification

Wider range of applications beyond protein analysis


Biomolecule Detection

Primarily detects proteins

Detects proteins and other biomolecules


Purpose in Research

Fundamental technique in molecular biology research

Used in diverse research areas, including genomics


Gel Staining

Typically not involved in gel staining

May include gel staining steps


Utility in Proteomics

Integral part of proteomic workflows

May be incorporated into proteomic experiments


Sample Preparation

Requires protein extraction and denaturation

Sample preparation may vary depending on the target



Analyzes protein size and quantity with high resolution

Resolution can vary depending on the method


Antibody Specificity

Requires highly specific antibodies

Requires specific antibodies for target molecule



Limited to protein analysis

More versatile but may have specific limitations


Protein Separation

Focuses on protein separation and identification

Separation may target various biomolecules


Protein Detection Sensitivity

Typically high sensitivity for protein detection

Sensitivity can vary depending on the application


Biomolecule Detection Sensitivity

May not be suitable for detecting non-protein biomolecules

May have sensitivity for diverse targets


Data Interpretation

Primarily interprets protein-related data

Data interpretation may encompass various targets


Transfer Efficiency

Efficiency can vary depending on the method used

Transfer efficiency may vary with the target


Method Variations

Western Blot is a specific method within immunoblotting

Immunoblotting includes various methods


Gel Type

Often used with polyacrylamide gels

May involve different gel types based on the target


Sample Size

Suitable for a wide range of sample sizes

Sample size may vary depending on the application


Commercial Kits Availability

Many commercial kits available for Western Blotting

Kits may vary depending on the specific technique


Specificity of Results

Typically specific to proteins

Specificity depends on the target biomolecule


Target Biomolecule Variability

Focuses on protein variability

Detects variability in different biomolecules


Data Analysis Tools

Software tools specific to Western Blot analysis

May require specific tools based on the target


Transfer Buffer Composition

Often uses Tris-glycine buffer

Buffer composition may vary with the target


Protein Denaturation

Involves protein denaturation with SDS

Denaturation conditions may vary based on target


Electrophoretic Mobility

Analyzes protein mobility based on size and charge

Mobility analysis may differ depending on the target


Historical Use

Developed as a protein-specific technique

Evolved to encompass a broader range of molecules


Detection Sensitivity Variation

Sensitivity can be optimized for proteins

Optimization may vary based on the target


Commonly Used in

Molecular biology and biochemistry research

Various scientific disciplines and applications


Gel Electrophoresis Parameters

Parameters are optimized for protein separation

Parameters may be adjusted for different targets


Subtypes or Variants

Various types such as 1D, 2D, and native Western Blots

Immunoblotting includes a wide range of variants


Clinical Diagnostics

Less commonly used for clinical diagnostics

May find applications in clinical diagnostics


Biomolecule Identification

Focuses on protein identification

Identifies a broader range of biomolecules


Antibody Validation

Requires validation of antibodies for specific proteins

Validation may differ based on the target molecule


Evolving Techniques

Continuously evolving with advancements in proteomics

Techniques may evolve based on specific applications


Cross-References in Literature

Often referred to as immunoblotting in recent literature

May still be referred to as Western Blotting in older literature

Frequently Asked Questions (FAQs)

Q1.Describe SDS-PAGE.

Proteins are separated using the SDS-PAGE (Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis) method according to their molecular weight. It involves moving proteins across a gel matrix in accordance with their size using an electric field.

Q2.What is the process of protein transfer (blotting)?

Proteins are moved from the gel to a membrane (often nitrocellulose or PVDF) after gel electrophoresis. Western blotting or electroblotting is a technique used to do this utilizing an electric field. The spatial layout of the transferred proteins on the gel is preserved.

Q3.In a Western blot, how is the protein signal picked up?

Enzymatic or fluorescent techniques are generally used to visualize the protein-antibody complexes. Enzymatic techniques require the use of enzyme-conjugated secondary antibodies and a substrate that, following enzymatic reaction, yields a visible result. Fluorescent techniques use antibodies that have been fluorescently marked.

Q4.Exist several types of immunoblotting?

Yes, there are variations like multiplex Western blotting, quantitative Western blotting, and reverse-phase protein arrays (RPPAs). These modifications aim to improve sensitivity, quantification accuracy, and the capacity for concurrent analysis of many targets.

Q5.What are immunoblotting's drawbacks?

Immunoblotting has some drawbacks, such as the potential for nonspecific antibody binding, variability in the efficiency of protein transfer, and the need for particular antibodies. Furthermore, the method might not reveal information on the localization of proteins within cells.

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