MRN Purification

Used in our papers:¹²³

Contents

1. Materials
2. Purification

1. Materials

1.1 Buffers

• MRN Lysis Buffer: 50 mM Potassium Phosphate (KH2PO4), 0.5 M KCl; 10% (vol/vol) glycerol; 25 mM imidazole; 20 mM b-mercaptoehtanol; 400 μL 50% Tween-20; 400 μL of phenylmethylsulfonyl fluoride (PMSF; 17 mg mL-1; AC215740500; Fisher Scientific)
• High-salt Ni2+ A Buffer: 50 mM Potassium Phosphate (KH2PO4), 0.5 M KCl; 10% (vol/vol) glycerol; 25 mM imidazole; 20 mM b-mercaptoethanol
• Low-salt Ni2+ A Buffer: 50 mM Potassium Phosphate (KH2PO4), 50 mM KCl; 10% (vol/vol) glycerol; 25 mM imidazole; 20 mM b-mercaptoethanol
• Low-salt Ni2+ B Buffer: 50 mM Potassium Phosphate (KH2PO4), 50 mM KCl; 10% (vol/vol) glycerol; 250 mM imidazole; 20 mM b-mercaptoehtanol
• A Buffer: 25 mM Tris·HCl pH 8.0, 100 mM NaCl, 10% (vol/vol) glycerol, 1 mM DTT
• 0-salt A Buffer: 25 mM Tris·HCl pH 8.0, 100 mM NaCl, 10% (vol/vol) glycerol, 1 mM DTT
• B Buffer: 25 mM Tris·HCl pH 8.0, 1 M NaCl, 10% (vol/vol) glycerol, 1 mM DTT

1.2 Resins and columns

• Ni2+-NTA resin (Qiagen; 30410)
• Anti-FLAG M2 resin (A2220; Sigma-Aldrich)
• Hi-Trap SP column (17115101; GE Healthcare)
• Superose 6 30/300 GL Increase column

2. Purification

1. Follow the above protocol for producing a pellet of insect cells expressing MRN from 60 15-cm dishes.
2. Quickly thaw the pellet and resuspend in 40 mL of MRN Lysis Buffer.
3. Homogenize pellet in a 50 mL Dounce homogenizer using an “A” piston.
4. Sonicate cells on ice three times for 30s each time with a 30-s recovery on ice.
5. Centrifuge the mixture at 35,000 rpm (Beckman Optima XE ultracentrifuge; 45Ti rotor) for 1 hr at 4°C.
6. Incubate supernatant with 10 mL of equilibrated Ni-NTA resin for 1 hr at 4°C with gentle agitation.
7. Centrifuge the sample at 1500 rpm for 3 minutes.
8. Remove the supernatant, add 40 mL of High-salt Ni2+ A buffer, and incubate at 4°C with gentle agitation for 5 minutes.
9. Centrifuge the sample at 1500 rpm for 3 minutes.
10. Remove the supernatant, add 40 mL of Low-salt Ni2+ A buffer, and incubate at 4°C with gentle agitation for 5 minutes.
11. Centrifuge the sample at 1500 rpm for 3 minutes.
12. Remove the supernatant, add 40 mL of 10% Low-salt Ni2+ B buffer (4 mL Ni2+ B + 36 mL Ni2+ A), and incubate at 4°C with gentle agitation for 5 minutes.
13. Centrifuge the sample at 1500 rpm for 3 minutes.
14. Add 15 mL of Ni2+ B buffer to resin and incubate for 30 minutes at 4°C.
15. Transfer Ni-NTA resin to disposable column and collect the elution.
16. Dilute the elution with 15 mL A Buffer and load onto a 50-mL superloop.
17. Equilibrate a Hi-Trap Q column on an FPLC with A and B buffers.
18. Load the sample onto the Q column using the superloop and wash until the UV stabilizes.
19. Elute with a step to 100% B buffer and collect 0.5 mL fractions.
20. Combine the fractions with the highest UV intensity (up to 5mL).
21. Keep a small aliquot for analysis. Dilute the prep in 0-salt A buffer until the NaCl concentration is less than 100mM.
22. Equilibrate a Hi-Trap SP column on an FPLC with A and B buffers.
23. Load the sample onto the SP column using the superloop and wash until the UV stabilizes.
24. Elute with a step to 100% B buffer and collect 0.5 mL fractions.
25. Equilibrate a Superose 6 30/300 GL Increase column on an FPLC with A and B buffers.
26. Take the highest concentration sample from SP and load it on the column using a 500 µL injection loop. Pool highest concentration fractions.
27. Aliquot sample, snap freeze with liquid nitrogen, and store at -80°C.

Footnotes

1. Myler et al. (2017). Single-molecule imaging reveals how Mre11-Rad50-Nbs1 initiates DNA break repair. Molecular Cell. ↩

2. Myler et al. (2019). Purification and Biophysical Characterization of the Mre11-Rad50-Nbs1 Complex. Methods Mol Biol. ↩

3. Soniat et al. (2023). The MRN complex and topoisomerase IIIa–RMI1/2 synchronize DNA resection motor proteins. Journal of Biological Chemistry. ↩