Nickel Bead Purification Amy Grace DuPage 1X BUFFER: (makes 50ml) 20mM imidazole, pH 8.0 0.5% Igepal 0.5mM DTT 1X PBS (2ml of 500mM imidazole stock) (1.5ml of 10% Igepal stock) (25µL of 1M DTT stock) (5ml of 10X PBS stock) HIGH SALT BUFFER: (makes 30mls) 350mM NaCl (1.8ml of 5M NaCl stock) 20nM imidazole (1.2ml of 500mM imidazole stock) 0.5mM DTT (15µL of 1M DTT stock) 1X PBS (3ml of 10X PBS stock) LOW SALT BUFFER: (makes 50ml) 20nM imidazole (2ml of 500mM imidazole stock) 0.5mM DTT (25µL of 1M DTT stock) 1X PBS (5ml of 10X PBS stock) ELUTION BUFFER: 300mM imidazole 1mM DTT 1X PBS Method: 1) Weigh out 10g lysed yeast. 2) Add 20µL each of pepstatin and aqueous inhibitors immediately. 3) Add equal volume 1X Buffer to yeast. 4) Once yeast starts to thaw, add 100µL PMSF (stock = 1:200) 5) Spin 80K rpm for 20 min. in TLA 100.3 rotor at 4C. (Each tube holds 3ml). **While spinning, wash 0.7 to 1ml bead mixture two times with 1X buffer. 6) Remove supernatant (approx. 2.5ml) and add to washed nickel beads in 15ml conical. Avoid white crud and interface. Save 50µL HSS for gel. 7) Fill up to 15ml with 1X buffer and rotate for 1 hour at 4C. **While rotating, prepare 30ml high salt buffer and 50ml low salt buffer without NP-40. 8) Centrifuge bead solution at 4˚C at 1.5 rpm. Save 50µL FT for gel. 9) Wash twice with 15ml of high salt buffer. 10) Wash three times with 15ml of low salt buffer. Decant supernatant and save 3µL beads for gel. 11) Add beads to a 0.5ml tube. Using a syringe, poke a small hole in the bottom of the tube. Place this tube inside a 1.5ml tube with cap cut off. Spin briefly at full speed until all liquid is removed from beads. 12) Change 1.5ml tubes and elute with 2 X 200µL elution buffer. Keep at 4˚C until ready for FPLC. FPLC Notes: Use Sup12 column and HEKG10 buffer + 1mM DTT.