Table 4. Kinetochore protein purification buffers.
| Protein | Purification buffers |
|---|---|
| Dam1c-FLAG |
Lysis: 50 mM sodium phosphate buffer pH 6.9, 500 mM NaCl, 1 mM PMSF, Roche protease inhibitor tablets FLAG wash: 50 mM sodium phosphate buffer pH 6.9, 500 mM NaCl, 1 mM PMSF, Roche protease inhibitor tablets FLAG elution: 50 mM sodium phosphate buffer pH 6.9, 500 mM NaCl, 1 mM PMSF, Roche protease inhibitor tablets, 200 ug/ml 3x FLAG peptide SEC: 50 mM sodium phosphate buffer pH 6.9, 500 mM NaCl |
| Mif2-MBP |
Lysis: 30 mM HEPES buffer pH 7.5, 2 M NaCl, 10% glycerol, 1 mM TCEP, 1 mM PMSF, Roche protease inhibitor tablets Amylose resin elution: 30 mM HEPES buffer pH 7.5, 100 mM NaCl, 10% glycerol, 1 mM TCEP, 1 mM PMSF, Roche protease inhibitor tablets, 10 mM maltose QA: 30 mM HEPES buffer pH 7.5, 100 mM NaCl, 10% glycerol, 1 mM TCEP, 1 mM PMSF, Roche protease inhibitor tablets QB: 30 mM HEPES buffer pH 7.5, 1 M NaCl, 10% glycerol, 1 mM TCEP |
| MIND-FLAG |
Lysis: 50 mM HEPES buffer pH 7.5, 200 mM NaCl, 10% glycerol, 0.5% NP40, 1 mM EDTA, 1 mM PMSF, Roche protease inhibitor tablets FLAG wash: 50 mM HEPES buffer pH 7.5, 200 mM NaCl, 10% glycerol, 1 mM EDTA, 1 mM PMSF, Roche protease inhibitor tablets FLAG elution: 50 mM HEPES buffer pH 7.5, 200 mM NaCl, 10% glycerol, 1 mM EDTA, 1 mM PMSF, Roche protease inhibitor tablets, 200 ug/ml 3x FLAG peptide SEC: 50 mM HEPES buffer pH 7.5, 200 mM NaCl, 1 mM EDTA |
| Ndc80c-FLAG |
Lysis: 50 mM HEPES buffer pH 7.6, 200 mM NaCl, 10% glycerol, 1 mM EDTA, 1 mM PMSF, Roche protease inhibitor tablets FLAG wash: 50 mM HEPES buffer pH 7.6, 200 mM NaCl, 10% glycerol, 1 mM EDTA, 1 mM PMSF, Roche protease inhibitor tablets FLAG elution: 50 mM HEPES buffer pH 7.6, 200 mM NaCl, 10% glycerol, 1 mM EDTA, 1 mM PMSF, Roche protease inhibitor tablets, 200 ug/ml 3x peptide SEC: 50 mM HEPES buffer pH 7.6, 200 mM NaCl, 1 mM EDTA |
| OA-FLAG |
Lysis: 50 mM HEPES buffer pH 7.5, 200 mM NaCl, 10% glycerol, 0.5% NP40, 1 mM PMSF, Roche protease inhibitor tablets Low salt FLAG wash: 50 mM HEPES buffer pH 7.5, 200 mM NaCl, 10% glycerol, 1 mM EDTA, 1 mM PMSF, Roche protease inhibitor tablets High salt FLAG wash: 50 mM HEPES buffer pH 7.5, 2 M NaCl, 10% glycerol, 1 mM EDTA, 1 mM PMSF, Roche protease inhibitor tablets FLAG elution: 50 mM HEPES buffer pH 7.5, 200 mM NaCl, 10% glycerol, 1 mM EDTA, 1 mM PMSF, Roche protease inhibitor tablets, 200 ug/ml 3x FLAG peptide SEC: 50 mM HEPES buffer pH 7.5, 200 mM NaCl, 1 mM EDTA |