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Western Blotting

Western Blotting

Western blotting is an analytical technique for protein identification. It has various applications in immunology and medicine, including detecting diseases like bovine spongiform encephalopathy, mad cow disease, and human and feline immunodeficiency virus from biological samples.
The technique begins with separating proteins from the sample using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by protein transfer, immunoblotting, and finally, protein detection.

Southern Blot

Southern Blot

Agarose gel electrophoresis is very useful in separating DNA fragments by size. Running a DNA ladder containing fragments of the known length alongside the sample helps determine the approximate length of the sample DNA fragments. However, additional steps are needed to verify the sequence identity of the sample DNA fragments.
Denatured DNA fragments must be transferred onto a carrier membrane from the gel to make it accessible to a probe - a small ssDNA fragment complementary to the target DNA...

Two-dimensional Gel Electrophoresis

Two-dimensional Gel Electrophoresis

Two-dimensional gel electrophoresis is a high-resolution protein separation method first introduced by O' Farrell and Klose in 1975. This method involves protein separation by two dimensions, mass and charge, making it more accurate than one-dimensional gel electrophoresis.
The first dimension separation uses the isoelectric focusing or IEF technique performed on immobilized pH gradient (IPG) strips that separate proteins according to their isoelectric points.
Biological samples, such...

SDS-PAGE

SDS-PAGE

Gel electrophoresis is a method that separates biological macromolecules like nucleic acids or proteins by forcing them to pass through a gel matrix under an electric field.
A variation of gel electrophoresis, termed polyacrylamide gel electrophoresis (PAGE), is commonly used for separating proteins according to their molecular size by passing them through a polyacrylamide gel. Because of the varying charges associated with amino acid side chains, PAGE can be used to separate intact...

Electrophoresis: Overview

Electrophoresis: Overview

Electrophoresis is a powerful analytical separation technique that relies on the differential migration of charged species when subjected to an electric field. The core strength of electrophoresis lies in its ability to separate high-molecular-weight species in complex mixtures. It has found widespread use in biochemistry, molecular biology, and analytical chemistry, allowing the separation of compounds like amino acids, nucleotides, carbohydrates, and proteins with excellent resolution.
There...

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Related Experiment Video

Updated: Oct 13, 2025

The Fastest Western in Town: A Contemporary Twist on the Classic Western Blot Analysis

The Fastest Western in Town: A Contemporary Twist on the Classic Western Blot Analysis

Published on: February 5, 2014

Western blotting.

Biji T Kurien¹, R Hal Scofield

¹Arthritis and Immunology Program, Oklahoma Medical Research Foundation, Oklahoma City, 73104, USA. biji-kurien@omrf.ouhsc.edu

Methods (San Diego, Calif.)

|February 18, 2006

Summary

This summary is machine-generated.

Western blotting, a key protein detection method, has evolved significantly since 1979. This review covers various protein transfer techniques, including diffusion, vacuum-assisted flow, and electrophoretic elution, for enhanced immunodetection.

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Last Updated: Oct 13, 2025

The Fastest Western in Town: A Contemporary Twist on the Classic Western Blot Analysis

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Published on: February 5, 2014

Western Blotting: Sample Preparation to Detection

Western Blotting: Sample Preparation to Detection

Published on: October 14, 2010

Western Blotting Using the Invitrogen NuPage Novex Bis Tris MiniGels

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Area of Science:

Biochemistry
Molecular Biology
Immunology

Background:

Western blotting (protein blotting or immunoblotting) is crucial for detecting low-abundance proteins post-electrophoresis.
The protein transfer step, established in 1979, has seen numerous advancements.
Diverse methods now exist for transferring proteins from gels to membranes.

Purpose of the Study:

To review and describe various protein transfer procedures used in Western blotting.
To compare methods based on diffusion, vacuum-assisted solvent flow, and electrophoretic elution.
To provide an overview of antigen detection methods on blots.

Main Methods:

Review of established and emerging protein transfer techniques.
Categorization of methods by underlying principles: simple diffusion, vacuum-assisted solvent flow, electrophoretic elution.
Summary of common antigen detection strategies for immunoblots.

Main Results:

Multiple methods exist for protein transfer, each with distinct mechanisms.
Technological evolution has provided researchers with varied options for optimizing protein blotting.
Effective antigen detection relies on appropriate post-transfer methodologies.

Conclusions:

Protein transfer in Western blotting has a rich history of innovation.
Researchers can select from diverse methods (diffusion, vacuum, electrophoresis) for protein transfer.
Understanding these transfer techniques is vital for successful protein immunodetection.