Protocolisfor150mlofcellculture. 1.Spindowncellsandwash1Xin10mlicecoldPBS. 2.ResUSPendpelletin20mllysisbuffer:50mMTrispH8.01%NP-401mMPMSF1µg/mlleupeptin 3.Transfertooakridgetubesandsonicateatasettingof4for20seconds. 4.SpinlysateinSS-34for20minutesat10,000rpm@4°C. 5.Bindsupernatantto0.5mlNi-NTApre-equilibratedinlysisbuffer.Rockat4°Cfor1hourtoovernight. 6.TransferslurrytoBio-Radeconocolumnandcollectflowthru. 7.Washwith20ml50mMTrispH8.0,500mMNaCl. 8.Washwith20ml50mMTrispH8.0,500mMNaCl,5mMimidazole. 9.Elutewith5ml50mMTrispH8.0,500mMNaCl,0.5Mimidazole.Collect1mlfractions. 10.Igenerallypoolfractions1and2anddialyzeovernightagainst2Lofdesiredbuffer.