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.
新型增塑剂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 l0脉动测速min at 4C: ; 高频磁芯(3) Wash cell with PBS;
(4) Centrifuge 1500rpm lOmin at 4 C ;
(5) Suspend cell in 5 xVolume-cell-weight CE buffer
投币器
(lOmM HEPES, 60mM KCI , ImM DTT , ImM分
合闸电磁铁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 lxNE buf-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 12000rpm Smin at 4 0C ;定位片
(14) Supematant is nuclear extract;
(15) Aliquot into tubes (5-10111 0f samples);
(16) Detect by SDS-PAGE and quantified.
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