真核表达纯化蛋白步骤

Purification of protein with 3xflag: Collect cells and wash with PBS, add 1ml lysis buffer (Tris 50mM, 500mM NaCl, 10% Glycerol, 0.5% NP40, 1mM DTT, 1mM EDTA, 1mM PMSF and protease inhibitor cocktail), put cells on ice for 30 min, vortex for 15s every 10min. Centrifuge for 10min at 20000xg.

Equilibrate 25ul M2 Flag Beads (sigma) with 1ml lysis buffer, incubate cell lysis buffer with Beads for more than 6 hours at 4℃.

Wash with lysis buffer 3 times.

Elute with 50ul 0.5mg/ml 3xFlag peptide (Genescript) for more than 12 hours at 4℃.

Prepare Cell Lysate -Shen Lab

ES cell lines (mWTFENI, mE160D)

(1) Trypsinize cell in the dish;

(2) Centrifuge 1 500rpm l0min at 4C: ;

(3) Wash cell with PBS;

(4) Centrifuge 1500rpmlOmin at 4 C ;

(5) Suspend cell in 5 xVolume-cell-weight CE buffer

(lOmM HEPES, 60mM KCI , ImM DTT , ImM

EDTA , proteinase inhibitor cockt-ail & fresh_0.075

NP-40);

(6) Incubate on ice for 10 min;

(7) Centrifuge 1500rpm lOmin at 40C, pellet is nuclei

part;

(8) Add SxVolume CE buffer (without NP-40) of

pellet weight (measure cells);

(9) 1500rpm Smin at 4C;

(IO) Resuspend in lxNEbuf-fer (25% glycerol,

20mM Tris-HCI, 420mM NaCI, 1.5mM MgCI2,

0.2mM EDTA and proteinase inhibitor cocktail);

(II) Vorrtex shortly;

(12) Incubate on ice for 60 min, vortex shortly during

incubation;

(13) Centrifuge 12000rpmSmin at 4 0C ;

(14) Supematant is nuclear extract;

(15) Aliquot into tubes (5-10111 0f samples);

(16) Detect by SDS-PAGE and quantified.