WIPI2d Expression and purification

Transfect 300mL of HEK GNTI cells

Dilute PEI with Warm Hybridoma-SFM(1X)

In a separate tube, dilute DNA with Hybridoma-SFM(1X)

Add PEI to DNA dilution. Incubate mixture for 0h 30m 0s at 37°C

Add mixture to cells. Let cells grow for 60h 0m 0s

Harvest Cells 2500rpm,4°C

Wash pellet with cold PBS. Store pellet until Puification.

Resuspended pellet in 25mL of lysis buffer (50mM Hepes pH 7.45, 300mM NaCl, 1mM TCEP, 1% Triton X, PI cocktail)

Let rock at4°C for 0h 20m 0s

Clarify lysate for 18000rpm,4°C

Rock supernatant with equilibrated strep resin for 2h 0m 0s at 4°C

Let supernatant FT gravity column

Wash with 50CV wash buffer (50mM Hepes pH 7.45, 300mM NaCl, 1mM TCEP)

Elute 2X with 5mL of wash buffer spiked with 5mM des-thiobiotin

Concentrate elution and inject onto pre-equilibrated S200 10/30 column (25mM Hepes pH 7.45, 150mM NaCl, 1mM TCEP)

Pool peak fractions, concentrate, snap freeze, and store at -80C