Davebrid: wrote initial protocol from Seraphin lab page

2010-05-24T20:16:46Z

wrote initial protocol from Seraphin lab page

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

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

TAP Purification of Yeast Extracts - Revision history

Davebrid: wrote initial protocol from Seraphin lab page