METHODS IN ENZYMOLOGY《DNA Repair Enzymes.Cell, Molecular, and Chemical Biology》(2017)
Contents
1. Introduction 2
2. Suspension Cell Growth and Handling for iPOND and aniPOND 5
2.1 Optimizing Growth Conditions to Obtain iPOND/aniPOND Cell Numbers 5
2.2 Handling of Suspension Cells During Pulse and Chase 7
3. Optimizations to the aniPOND Protocol to Increase Functionality 7
3.1 Optimized Sonication Regimen 9
3.2 Preventing Chromatin Precipitation in Sonicated Chromatin 11
3.3 Eliminating Sources of Background 13
3.4 Optimization Limitations 15
4. Comparison of iPOND and aniPOND in Suspension Cells 16
5. Protocol for Optimized aniPOND in Suspension Cells 18
5.1 Suspension Cell Growth to aniPOND Experimental Numbers 19
5.2 EdU Pulse, Thymidine Chase, and Click Reaction 20
5.3 Solubilization and Pulldown of Biotin-Labeled Chromatin 24
5.4 Protein Elution and Quantitative Western Analysis 27
5.5 Troubleshooting 30……
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