xiap????????????

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xiap????????????
??????
??09-06-01
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• ??
• ?????
• ??
• ??

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??

• X????????(X-linked inhibitor of apoptosis
  protein,XIAP)??????????????,???????caspase??,?????
  ??????????????????????????????????????????????????
  ????????????????????
• ????pET151-XIAP??????????,Ni-NTA????????
• ???????(??OD600 0.6???30?IPTG??0.5ug/ml?????4h?)?
• ?????,??????pH????(pH 5.4?pH 4.9)?

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XIAP????????
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?????

• ??
• ?????pET151-XIAP???JMY109?Rosseta??????
• ????
• ????LB??????????-ß-??????(IPTG)
• ???????Tris?NaH2PO4
• SDS-PAGE??30????? 1.5mol/L Tris(pH8.8)?10SDS?
  10???? ?TEMED ?1mol/L Tris(pH6.8)

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???????
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• pET151??????
• ??????
• 1.???????(CaCl2) 2.??
• ??????(IPTG??????OD?????)
• ????????
• 1.Ni-NTA??????? 2.??

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????????
1.??OD????
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• ???????,?OD?0.6?,?????????????OD??????,???????
  ?,???????????37?,IPTG??5ug/ml,????4h????,????OD??0
  .6-1.0????,???????????

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2.????
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• OD??0.6,IPTG???IPTG??5ug/ml,????4h????
  ,30??????????????,??????????????,?????????,???????
  ???

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3. IPTG??
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• ??30?,??OD600 0.6,????4h?????IPTG????
  0.5ug/ml??????????,???0.5ug/ml?,????????,????IPTG?
  ????,????????????????????IPTG???0.5ug/ml??????????
  ???????????????????

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4.????
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• ??30?,??OD600 0.6,IPTG??0.5ug/ml?,????
  ???????????,?????4-8h?,????????,??????????????10h?
  ,?????????????????????????????

?? ????4???,??????,??30?,??OD600
0.6,IPTG??0.5ug/ml,????4h,???XIAP?????????????????
??
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??????????
Ni-NTA???????????
MMarker A???? B??? W???
5.9????pH 4.5????pH
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Ni-NTA????????SDS-PAGE???
MMarker A???? B??? W???
5.9????pH 4.5????pH
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Ni-NTA????????SDS-PAGE???(?????PH)
MMarker A???? B??? W??? 5.9????pH 4.5????pH
5.4?4.9???pH
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• ?????????pH????,??????pH?4.9?4.5?????????????
  ???????????????????,???????His????,???????????????
  ????,????????pH?????

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??

• 1.??????????????,????????????????????????????,????
  ???????????,????????,???????,????
• 2.????????????(1)??????? (2)???????(3)????(4)???
  ??????
• 3.???????????
• 4.?????????????

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??

• 1. ????????????????OD600 0.6???30?IPTG??0.5ug/ml?
  ????4h?
• 2. ??????,??????pH????,?????????????????

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Thanks!
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Caspases

• ??????????????????????,???????????????????????????
  
• ???????????????,??????????(????1?)???,????????????
  ,?????????????,????????,?????????????

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Caspases

• ????????11?caspase
• ??caspases?,caspase 1?caspase
  11,??????caspase 4???????????????,???????????????
  caspase 2,caspase 8,caspase 9?caspase
  10????????????????????caspase 3,caspase
  6?caspase 7,??caspase 3?7??????????????,????PARP,D
  FF-45(DNA fragmentation factor-45),??DNA????????DN
  A?????caspase-6????lamin A?keratin
  18,???????????????????
• ??????caspase???????????,?????????????