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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 as cells...

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 proteins...

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: Jun 4, 2026

Western Blotting: Sample Preparation to Detection

Western Blotting: Sample Preparation to Detection

Published on: October 14, 2010

Western blotting.

N R Madamanchi¹, M S Runge

¹Division of Cardiology, University of Texas Medical Branch, Galveston, TX.

Methods in Molecular Medicine

|February 19, 2011

Summary

This summary is machine-generated.

Western blotting rapidly detects and quantifies specific proteins using antibody-based methods. This sensitive technique, employing enzyme-linked immunodetection, allows for precise protein identification from complex biological samples.

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Last Updated: Jun 4, 2026

Western Blotting: Sample Preparation to Detection

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Published on: October 14, 2010

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

Published on: February 5, 2014

Area of Science:

Biochemistry
Molecular Biology
Immunology

Background:

Western blotting is a key technique for detecting proteins and macromolecules.
It allows for the identification and quantification of specific proteins within complex mixtures.
The method involves immobilizing molecules onto nitrocellulose membranes for analysis.

Purpose of the Study:

To detail the methodology and applications of Western blotting.
To highlight the sensitivity and specificity of protein detection.
To explain the principles of antigen-antibody complex detection.

Main Methods:

Proteins are separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Separated polypeptides are transferred to a nitrocellulose membrane.
Detection involves incubation with primary antibodies, followed by labeled secondary antibodies (e.g., HRP or AP) and enhanced chemiluminescence (ECL).

Main Results:

The method enables sensitive detection of specific proteins.
Alkaline phosphatase (AP)-conjugated antibodies can detect 10-50 ng of protein.
Horseradish peroxidase (HRP)-conjugated antibodies can detect 0.5-1.0 ng of protein.

Conclusions:

Western blotting is a versatile and sensitive technique for protein analysis.
The choice of enzyme conjugate (AP or HRP) influences detection sensitivity.
High-affinity primary antibodies are crucial for optimal results.