Difference between revisions of "TAP Purification of Yeast Extracts"

Latest revision as of 20:16, 24 May 2010

Materials

• TAP Lysis Buffer (10 mM Tris, 150 mM NaCl, 0.1% NP40; make 100 mL, take from this to make other buffers). For other buffers add bold ingredients
• TEV Cleavage Buffer (10 mM Tris, 150 mM NaCl, 0.1% NP40, 0.5mM EDTA, 1 mM DTT; make 15 mL)
• CaM Binding Buffer (10 mM Tris, 150 mM NaCl, 0.1% NP40, 10 mM B-ME, 1 mM MgAc, 1 mM Imidazole, 2 mM CaCl2; make 40 mL)
• CaM Elution Buffer (10 mM Tris, 150 mM NaCl, 0.1% NP40,10 mM B-ME, 1 mM MgAc, 1 mM Imidazole, 2 mM EGTA; make 5 mL)
• Small BioRad Column
• IgG Sepharose
• Calmodulin Sepharose
• TEV Protease

Protocol

1. Resuspend 2L of yeast in 10 mL of water with a protease inhibitor tablet and french press.
2. Add 100 uL 1M Tris (10 mM), 250 uL NaCl (final 150 mM), 100 uL 10% NP40 (final 0.1%).
3. Centrifuge for 20 min at 50 000 RPM to clarify. Save a lysate sample.
4. Add 200 uL IgG sepharose beads for 2h at 4C.
5. Pour into column and wash with 30 mL of TEV Lysis Buffer.
6. Wash with 10 mL TEV Cleavage Buffer.
7. Close column, add 1 mL TEV Cleavage Buffer and add 100U of TEV, rotate 2h at 16C to digest.
8. Recover Eluate and wash with an extra 200 uL TEV Cleavage Buffer. Save a IgG sample
9. Add 3 mL CaM Binding Buffer and 3 uL of CaCl2 to titrate out the EDTA.
10. Add to this mixture 200 uL CaM Sepharose
11. Rotate for 1h at 4C
12. Wash with 30 mL Binding Buffer
13. Elute with CaM Elution Buffer, collecting 200 uL Fractions.

Source: http://www.embl.de/ExternalInfo/seraphin/TAPpurification.html