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Updated: Aug 8, 2026
Analysis of Cell Cycle Position in Mammalian Cells
Published on: January 21, 2012
Preliminary characterization of a monoclonal antibody (AS-2) against cell cycle related proteins
S Nigro1, A Rapallo, A Di Vinci
1Laboratory of Biophysics and Cytometry, National Institute for Cancer Research, Genoa, Italy.
Insights
A new monoclonal antibody, AS-2, identifies cell cycle-related proteins in human cells. These proteins show varying levels throughout the cell cycle and appear to associate with centrosomes during mitosis.
Area of Science:
- Cell Biology
- Immunology
- Molecular Biology
Background:
- Monoclonal antibodies are crucial tools for identifying specific proteins.
- Understanding cell cycle regulation involves identifying key protein markers.
Purpose of the Study:
- To describe a novel monoclonal antibody (AS-2) targeting proteins in human erythroleukemia cells.
- To characterize the cell cycle-related proteins recognized by AS-2.
Main Methods:
- Generation of a monoclonal antibody (AS-2) using isolated nuclei from human erythroleukemia cells.
- Immunodetection of target proteins in cytoplasm and nucleus.
- Flow cytometry analysis of protein content relative to DNA content.
- Cell sorting based on DNA content and AS-2 fluorescence.
Main Results:
- AS-2 (IgM type) recognized 200 kDa proteins in cytoplasm and 70/60 kDa proteins in nucleus.
- Protein levels were highest in late-G1, S, and G2 phases, lowest in M and early-G1.
- Interphase cells/nuclei showed dispersed staining; mitotic cells displayed two distinct dots.
- Preliminary data suggest cell cycle association and potential centrosome localization in mitosis.
Conclusions:
- The AS-2 antibody recognizes cell cycle-dependent proteins.
- These proteins may play a role in cell cycle progression and mitosis.
Abstract:
A monoclonal antibody (AS-2) raised by using isolated nuclei from a human erythroleukemia cell line as immunogen is described. AS-2 was of IgM type and recognized proteins present in both isolated cytoplasms and nuclei. The molecular weight of the AS-2 recognized proteins in the cytoplasm was 200 kDa and 70 and 60 kDa in the nucleus. The relative amount of these proteins were measured simultaneously with DNA content by flow cytometry. We found the highest protein content (or stainability) for both cells and nuclei in late-G1, S and G2, at approximately the same level, and the lowest content in M and early-G1. Sorting based on DNA content and AS-2 associated fluorescence helped identifying the staining pattern of cells and nuclei. Interphase isolated nuclei and cell cytoplasms were characterized by interdispersed staining over the entire surfaces while mitoses showed two dots only. The present preliminary data indicate that the proteins recognized by the AS-2 monoclonal are cell cycle related and suggest that in mitoses they are associated with the centrosomes.
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