Related Experiment Video

Updated: Aug 9, 2026

Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells
13:58

Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells

Published on: October 22, 2012

Nuclear and mitotically enhanced epitope

J C Hoffman1, J M Mullins

  • 1Department of Biology, Catholic University of America, Washington, DC 20064.

Cell Motility and the Cytoskeleton
|January 1, 1990
PubMed

Insights

Monoclonal antibody C9 identifies a novel nuclear antigen present throughout the cell cycle, becoming exposed during mitosis. This antigen localizes to centrosomes, kinetochores, and the midbody during cell division.

Area of Science:

  • Cell Biology
  • Immunology
  • Molecular Biology

Background:

  • Mitosis involves dynamic changes in protein localization and function.
  • Understanding cell cycle regulation requires identifying key regulatory proteins.
  • Centrosomes, kinetochores, and the midbody are crucial structures during cell division.

Purpose of the Study:

  • To develop and characterize a novel monoclonal antibody (MAb) targeting mitotic-specific antigens.
  • To investigate the cell cycle-dependent localization and molecular identity of a newly identified nuclear antigen.

Main Methods:

  • Hybridoma production using salt-extracted proteins from taxol-stabilized microtubules.
  • Immunofluorescence microscopy for antigen localization.
  • Cell permeabilization and detergent extraction for antigen accessibility.
  • Immunoblot analysis for polypeptide identification across species.

Main Results:

  • Monoclonal antibody C9 recognized an epitope on an antigen localized to interphase centrosomes and nuclei.
  • The nuclear antigen's availability was cell cycle-dependent but present throughout the cycle upon permeabilization.
  • The antigen was exposed during prophase, released into the cytoplasm during nuclear envelope breakdown, and re-accumulated at daughter nuclei.
  • Mitotic cytoplasmic antigens localized to centrosomes, kinetochores, and the midbody.
  • Immunoblot analysis identified a ~250 kD polypeptide for the nuclear antigen and 107/117 kD and ~250 kD polypeptides for mitotic cytoplasmic antigens.

Conclusions:

  • Monoclonal antibody C9 identifies a novel antigen with dynamic cell cycle-dependent localization.
  • This antigen plays a role in mitosis, associating with key structures like centrosomes and kinetochores.
  • The findings contribute to the understanding of nuclear and mitotic antigens and their roles in cell division.

More Related Videos

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes07:59

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Published on: March 25, 2014

Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope08:09

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

Published on: March 24, 2017

Related Concept Videos

Nuclear Protein Sorting01:34

Nuclear Protein Sorting

Nuclear protein sorting is the selective trafficking of histones, polymerases, gene regulatory proteins into the nucleus and exporting RNAs and ribosomes to the cytosol. It is a tightly controlled process that regulates gene expression within a cell.
Proteins targeted to the nucleus carry nuclear localization signals or NLS recognized by import receptors in the cytosol. Similarly, proteins with nuclear export signals are recognized by export receptors. Import and export receptors are...
Nuclear Localization Signals and Import01:46

Nuclear Localization Signals and Import

Proteins targeted to the nucleus carry short stretches of amino acid sequences called the nuclear localization signal or NLS. Classical nuclear localization signals are of two types: monopartite and bipartite NLS. Monopartite classical NLS (cNLS) consists of a single cluster of 4-8 amino acids. Bipartite cNLS consists of two clusters of  2-3 amino acids and a 9-12 residue long proline-rich linker bridging the two clusters. Signal clusters are rich in positively charged amino acids such as...
Nuclear Export01:42

Nuclear Export

The nucleus restricts several proteins within and allows others to pass. The restricted proteins possess a nuclear retention sequence or NRS, anchoring them to the nuclear lamins and preventing their transport to the cytosol. The non-restricted proteins, after their synthesis, are transported to their site of action, such as the cytosol or other organelles, with the help of nuclear export signals or NES.
NES are of three types- the canonical 10-residue long leucine-rich signal and other...
Hybridoma Technology01:31

Hybridoma Technology

Hybridoma technology is used for the large-scale production of monoclonal antibodies. Monoclonal antibodies bind to only a single antigenic determinant or epitope. Such antibodies are used in research, diagnostics, and disease therapy. The hybridoma technology established in 1975 by Georges Köhler and Cesar Milstein was awarded the Nobel Prize in Medicine in 1984 for revolutionizing research and therapy.
Hybridoma Selection
Commonly used fusion techniques — electroporation, polyethylene glycol...
Tagging and Fusion Proteins01:24

Tagging and Fusion Proteins

Proteins are involved in several cellular processes and biochemical reactions. Analyzing a specific protein of interest requires it to be isolated from the other proteins in the cell. This is achieved by overexpressing the specific gene in a suitable host to produce large quantities of the target protein. A tag or label is recombined with the gene to produce a fusion protein containing the target protein and the tag. The tags on these fusion proteins can then be used for easy detection and...
Immunogold Electron Microscopy01:20

Immunogold Electron Microscopy

Immunoelectron microscopy utilizes immunogold labeling of endogenous proteins with specific antibodies to detect and localize these proteins in cells and tissues. The procedure provides insights into the distribution and quantification of protein under different stimulation conditions offering clues about their functions. Conjugating highly electron-dense gold particles with primary or secondary antibodies allow antigen detection on and within cells, with high resolution and specificity.