membrane protein purification - how to pufify cd3 protein from jurkat cell (Feb/16/2002 )
####. I need your help.
Several weeks ago I tried to purify cd3 protein from jurkat cell. I washed the cell three times by PBS(pH7.4) and suspended the cell in 10ml PBS. Then I freeze and thrawn the cell four times. After centrifuged the mixture at 14000rpm for 90min, I dissolved the pricipition in 5ml 1%NP40,5mMNaPO4 pH7.5(in fact most of the recipication could not be dissolved). Then I dialysed the solution in PBS overnight. All the procediure was carried out in 4 centigrade.
But when I used the purified protein to do ELISA, I found it had no activity. Who could please tell me an efficient purification method? Thanks a lot.
have you tried briefly sonicating the sample?