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Optimization of Native and Formaldehyde iPOND Techniques for Use in Suspension Cells
Nathaniel E Wiest1, Alan E Tomkinson1
1University of New Mexico School of Medicine, Albuquerque, NM, United States.
Methods in Enzymology
|June 25, 2017
Summary
This study optimizes the isolation of proteins on nascent DNA (iPOND) technique for suspension cells. Modifications improve sample recovery and signal for studying DNA replication proteins.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- The isolation of proteins on nascent DNA (iPOND) technique enables the study of proteins interacting with replicating DNA.
- Existing iPOND protocols are primarily optimized for adherent mammalian cell lines.
Purpose of the Study:
- To adapt and optimize the iPOND and accelerated native iPOND (aniPOND) protocols for use with suspension cell lines.
- To directly compare the iPOND and aniPOND methods in suspension cells.
Main Methods:
- Modified cell culture, pulse, and chase conditions for suspension cells.
- Introduced key improvements to the aniPOND protocol to reduce sample loss and increase signal-to-noise ratio.
- Direct quantitative comparison of iPOND and aniPOND protocols.
Main Results:
- Successfully adapted both iPOND and aniPOND protocols for suspension cell lines.
- Optimized aniPOND protocol demonstrates reduced sample loss and enhanced signal.
- Quantitative comparison highlights the strengths and limitations of both iPOND and aniPOND.
Conclusions:
- The optimized aniPOND protocol provides a robust method for studying proteins associated with nascent DNA in suspension cells.
- This work expands the utility of iPOND techniques for a broader range of cell culture models.
- Detailed protocol provided for researchers using suspension cell lines.
