Flag-His purification mammalian cells

Flag His purification from mammalian cells. D. Cortez 8-10-05

Harvest and wash cells with PBS. Weigh the cells and determine approximate volume. Snap freeze with liquid nitrogen and store in –80.

Lysis

Lysis buffer: 50mM NaP pH=8

300mM NaCl

10% glycerol

0.5% triton X-100

5ug/ml aprotinin

5ug/ml leupeptin

1mM NaVanadate

1mM NaF

1mM PMSF

Volume = 10x weight of pellet (ie use 15ml for a 1.5g pellet)

Incubate on ice for 15min

Spin at approximately 14-17,000 x g for 10 min 4 degrees.

Binding to His Select beads (Sigma)

Pre-equilibrate 1ml of beads (1:1 v/v so actually approximately 0.5ml of packed beads) per 15ml of lysate with lysis buffer (wash 2x).

Incubate beads with lysate for 20min room temperature with rocking or rotating.

Wash beads 4x with Wash buffer: 50mM NaP pH=8

300mM NaCl

10% glycerol

0.5% triton X-100

5mM imidazole

1ug/ml aprotinin

1ug/ml leupeptin

0.1 mM NaVanadate

0.1 mM NaF

0.5 mM PMSF

Elute 3x with Elution buffer: Elution buffer is Wash buffer + 250mM Imidazole

I use 2ml of elution buffer each time (if using 15ml of original lysate) and I incubate the beads with the elution buffer for 3-5min per elution.

Combine eluate. Add equal volume of: 50mM NaP pH=8

10% glycerol

0.1% triton X-100

Also wash His Select beads to reuse – wash 1x with water, then 1x with 5ml of 6M guanidine Hcl for 20 minutes then wash with water again 1x. Column can now be reequilibrated if used immediately or stored by washing 1x in 30% ethanol and stored in 30% Ethanol at 4 degrees.

Binding to Flag Beads

Pre-equilibrate 500ul of beads (1:1 v/v so actually approximately 250ul of packed beads) by washing 1x with Flag wash buffer: 50 mM NaP pH=8

150 mM NaCl

10% glycerol

0.25% triton X-100

0.5 ug/ml aprotinin

0.5 ug/ml leupeptin

0.1 mM NaVanadate

0.1 mM NaF

0.1 mM PMSF

Then wash 1x in 0.1M glycine pH=3.5 for 2-3 minutes

Then wash 2x in Flag wash buffer.

Add lysate eluate to beads and incubate at 4 degrees rotating for 1 hour.

Wash beads 2x with Flag wash buffer.

Wash beads 2x with Flag wash buffer without any phosphatase or protease inhibitors.

Elution: Use 1.25ml of 0.1M glycine pH=3.5, 5 min room temperature; repeat with 1.0ml of 0.1M glycine pH=3.5, 3 min room temperature; repeat again with 0.75ml of 0.1M glycine pH=3.5, 1 min room temperature. After first elution add 125ul of 0.5M TrispH=7.5, 1.5M NaCl to neturalize the eluate; add 100ul to neutralize the second eluate; add 75ul to neutralize third eluate.

Combine all eluates. Spin at 1,000 x g for 5 minutes.

Split the eluate into three eppendorf tubes. Approximately 1ml per tube. Add 250ul of TCA to each sample. Incubate on ice for 10min. Spin at full speed in cold room 5 minutes.

Carefully remove supernatant with pipet (do not distub pellet which may be difficult to see).

Wash pellet by adding 400ul of ice cold acetone, spin again for 5 minutes. Remove acetone with pipet tip and repeat wash. By this point the precipitate should be visible. Dry precipitate using speed vac (approximately 10 minutes). Redissolve the precipitated proteins in SDS sample buffer – you probably will need to add 1-2 ul of 1M Tris pH=8.0 to neutralize the remaining acid. (If the dye is not blue then add the Tris, if it is blue then the pH is probably OK.) I have been adding 15ul 2X SB/tube.

Vortex to dissolve the proteins and incubate at 75 degrees for 10 minutes before loading onto SDS-PAGE gels.