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Related Concept Videos

Peptide Identification Using Tandem Mass Spectrometry01:33

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: May 25, 2026

A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation
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Verification of 2A peptide cleavage.

Andrea L Szymczak-Workman, Kate M Vignali, Dario A A Vignali

    Cold Spring Harbor Protocols
    |February 4, 2012
    PubMed
    Summary

    This protocol details assessing 2A peptide cleavage in multicistronic vectors using 293T cells and western blot analysis. This method ensures efficient, stoichiometric protein production for multigene delivery in biomedical research.

    Area of Science:

    • Biomedical technology
    • Molecular biology
    • Gene expression

    Background:

    • Multicistronic vectors are crucial for delivering multiple genes simultaneously.
    • 2A peptide sequences enable stoichiometric protein production from a single open reading frame (ORF).
    • Efficient assessment of 2A cleavage is vital for validating vector functionality.

    Purpose of the Study:

    • To provide a reliable protocol for assessing 2A peptide cleavage.
    • To demonstrate the utility of 293T cells for this assessment.
    • To enable efficient verification of multicistronic vector performance.

    Main Methods:

    • Transient transfection of 293T cells with 2A peptide-linked multicistronic vectors.
    • Western blot analysis to detect protein products.

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  • Utilizing antibodies against target proteins or anti-2A serum for detection.

    • Main Results:

    • Successful demonstration of 2A cleavage assessment in 293T cells.
    • Validation of western blot as an effective detection method.
    • Confirmation of stoichiometric protein production.

    Conclusions:

    • Transient transfection of 293T cells followed by western blot is an effective method for assessing 2A cleavage.
    • This protocol facilitates the development of reliable multicistronic vectors for multigene delivery.
    • The findings support the advancement of gene therapy and protein production technologies.