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Finkelstein Lab Wiki

BLM Purification

Used in our papers:123

Contents

Section titled “Contents”
  1. Materials
  2. Purification

1. Materials

Section titled “1. Materials”
  • BLM 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)
  • Ni2+ A Buffer: 50 mM Potassium Phosphate (KH2PO4), 0.5 M KCl; 10% (vol/vol) glycerol; 25 mM imidazole; 20 mM b-mercaptoethanol
  • Ni2+ A Buffer: 50 mM Potassium Phosphate (KH2PO4), 0.5 M 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
  • B Buffer: 25 mM Tris·HCl pH 8.0, 1 M NaCl, 10% (vol/vol) glycerol, 1 mM DTT
  • Ni2+-NTA resin (Qiagen; 30410)
  • Anti-FLAG M2 resin (A2220; Sigma-Aldrich)
  • Hi-Trap SP column (17115101; GE Healthcare)

2. Purification

Section titled “2. Purification”
  1. Grow Sf21 insect cells in 60 15-cm dishes with a baculovirus harboring FLAG-BLM-His6, as recommended by the manufacturer. Harvest 2 L of Sf21 cells 72 hours after infection. Centrifuge at 4,000 x g to pellet the cells, snap freeze in liquid nitrogen, and store at -80°C until purification.
  2. Thaw the pellet quickly at room temperature.
  3. Homogenize pellet in 40 mL of BLM Lysis Buffer in a Dounce homogenizer.
  4. Sonicate cells on ice three times for 30 s 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 to obtain soluble extract. Keep a small aliquot for analysis.
  6. Incubate supernatant with 8-10 mL of equilibrated Ni-NTA resin with Ni2+ A Buffer for 1 hr at 4°C with gentle rocking.
  7. Centrifuge the sample at 1,500 rpm for 2 minutes (Beckman Avanti J-26XP; JLA-16.250 rotor). Keep a small aliquot for analysis.
  8. Transfer Ni-NTA resin to a disposable column.
  9. Wash the resin with 50 mL of BLM Lysis Buffer three times.
  10. Add 15 mL of Ni2+ B Buffer: to resin and incubate for 30 minutes at 4°C.
  11. Elute the sample from the Ni-NTA resin. Keep a small aliquot for analysis.
  12. Add sample to 0.8 mL of equilibrated anti-FLAG M2 affinity resin and incubate for 30 minutes at 4°C.
  13. Transfer FLAG resin to a disposable column.
  14. Wash the resin with 15 mL of A buffer three times.
  15. Add 5 mL of A buffer + 100 μL of 3xFLAG peptide (5 mg mL-1) and incubate for 30 minutes at 4°C.
  16. Elute sample from FLAG resin. Keep a small aliquot for analysis.
  17. Run sample through a 1mL Hi-Trap SP using A Buffer and B Buffer. Perform a linear gradient over 10 CVs to elute protein.
  18. Pool aliquots containing BLM from the SP column and dialyze overnight in A Buffer.
  19. Determine proteins concentration using Bradford assay or BSA.
  20. Aliquot sample and snap freeze with liquid nitrogen and store at -80°C.
  21. The typical yield from 2 L of Sf21 cells is ~75-100 μg of ~300-400 nM protein.

Footnotes

Section titled “Footnotes”

  1. Soniat et al. (2019). Assembling the Human Resectosome on DNA Curtains. Methods Mol Biol. ↩

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

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