Phosphorylated RPA Purification

Used in our paper:¹

Contents

1. Materials
2. Protocol

1. Materials

• Buffer A-25mM Tris-HCl pH 8, 100mM NaCl, 10% glycerol
• HEK293T cells (Harvested at 70-80% confluency-grown 3-4 days in DMEM)

2. Protocol

1. Add 500 uL of Buffer A to 2-4 dishes of HEK293T cells (15 mL/dish).
2. Sonicate at 10-15% Amplitude for 30 seconds then put on ice for 1 min (Repeat 3x).
3. Clarify the lysate by centrifugation at 20,000xg for 10 minutes.
4. Determine concentration of extract via Bradford assay and adjusted to 10 mg mL-1.
5. Combine in a 50µL solution.
   1. 20 µL Buffer (final concentrations: 40mM HEPES pH 7.5, 8mM MgCl2, 0.5mM DTT, 3mM ATP, 100 ng of pcDNA3 plasmid DNA (an SV40 replication origin containing plasmid)
   2. 20 µL of 2-5 mg mL-1 HEK293T extract (final concentration: 0.8-2-5 mg mL-1)
   3. 10 µL 10 µM purified RPA (final concentration: 2 µM).
6. Incubated at 37˚C for 2 hours.
7. Purify from the extract by using a 1 mL HiTrap Q.
8. Assay by western blot using antibodies for pRPA at S4/S8, T21, and S33.

Footnotes

1. Soniat et al. (2019). RPA phosphorylation inhibits DNA resection. Molecular Cell. ↩