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Related Concept Videos

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.

Peptide Identification Using Tandem Mass Spectrometry

Peptide Identification Using Tandem Mass Spectrometry

Tandem mass spectrometry, also known as MS/MS or MS2, is an analytical technique that employs two mass analyzers. Essentially it is a series of mass spectrometers that helps isolate a particular biomolecule and then helps study its chemical properties.
This technique helps gather information regarding the protein from which the peptide was obtained and to study the peptides’ amino acid sequence. Identifying peptides from a complex mixture is an important component of the growing field of...

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Related Experiment Video

Updated: Jun 21, 2025

Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope

Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope

Published on: March 24, 2017

Sequential Double Immunoblotting with Peptide Antibodies.

Nicole Hartwig Trier¹, Gunnar Houen²

¹Department of Neurology and Institute of Translational Research, Rigshospitalet, Glostrup, Denmark. nicole.hartwig.trier@regionh.dk.

Methods in Molecular Biology (Clifton, N.J.)

|July 12, 2024

Summary

Western blotting, a protein detection method, uses SDS-PAGE and peptide antibodies for characterization. This study details sequential detection of protein forms, exemplified by calreticulin antibody specificity.

Keywords:

Calreticulin Double immunoblotting Peptide antibodies

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Last Updated: Jun 21, 2025

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Published on: March 24, 2017

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Area of Science:

Biochemistry
Molecular Biology
Immunology

Background:

Immunoblotting, or western blotting, is crucial for protein detection and characterization.
Combining SDS-PAGE with immunoblotting and specific antibodies enhances protein analysis.
Understanding protein variations is vital for cellular and disease mechanism studies.

Purpose of the Study:

To present a protocol for sequential detection of diverse protein forms using peptide antibodies.
To exemplify this method by characterizing antibody specificity for calreticulin variants.

Main Methods:

Utilizing sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE) for high-resolution protein separation.
Employing immunoblotting onto synthetic membranes for protein transfer.
Implementing sequential detection with highly specific peptide antibodies.

Main Results:

Demonstrated a protocol for sequential protein form detection.
Characterized antibody specificity for different forms of the protein calreticulin.
Validated the utility of double SDS-PAGE immunoblotting for protein analysis.

Conclusions:

The described protocol enables precise characterization of individual proteins and their variants.
This method is valuable for studying protein roles in cellular processes and disease.
Peptide antibodies are effective tools for specific protein form detection.