Western Blot??

1
???

• ??????????
• ??????

2

• Western Blot??
• Western Blot????
• Western Blot??????

3
Western Blot ??

• ???(blotting)?????????????,??????????????????????
• 1975?,Southern????DNA?????????(NC?)?,???DNA-RNA???
  ????DNA?????,??Southern????
• ??????????,?RNA??????????,?RNA???????Northern???,?
  ??????????????????Western???,??????????????????Eas
  tern????

4
Western Blot ????
5
Western Blot ??

• ?????????
• ???????-????
• 1-5ng????????

6
Western Blot??

• ???????????
• ????????????
• ?????????
• ??????????

7
Western Blot ??

• ???????
• SDS-PAGE
• ??
• ??
• ????
• ????
• ????

8
???????

• ???????????????
• ??????
• ????????????????????????????????????????????
  ???????????? ?
• ???????
• ????????????????????????,????????????
• ???????????????

9
???????

• ????????????????Bradford?(??????)?Lowry?(Folin-??
  ??)? BCA?????????,??????????????????????????????,?
  ??????
• ??????????????
• http//bbs.bbioo.com/thread-23639-1-1.html

10
???????

• 2SDS-PAGE?????
• DTT?????14??????,????????,-20????
• ?11??????????,95100???515min,????????,??????20-
  25µl,????2050µg?

1M Tris-HCl(pH6.8) 10 ml
1M DTT 20 ml
SDS 4 g
?? 20 ml
??? 0.2 g
??? 100ml
11
SDS

• ?????? ???
• ??????SDS??????????????-SDS???
• ???-SDS????????????????????????,????
  ?????????????
• ????????????????????,??????????

12
???-SDS?????
(1)???????,?????????(2)??1g ?????1.4g
SDS (3)???????????-SDS????????? (4)???????????????
???
13
SDS?????????

• ???????????
• SDS????????????,??????????????,???????????????

14
?SDS-PAGE?????

• SDS-PAGE ??????????SDS?????????????,SDS???????????
  ???,???????????????,????????????????
• ????????(??????,DTT)??????????????????????????????
  ??????????????SDS??????????????-SDS????
• ???????SDS????,?????????? ?????????,?????????????
  ?
• ???????????????????????? ???????,?????????????

15
????

• ?????N,N-??????? ??
• SDS
• ???Tris???
• TEMED
• ????
• Tris-????????

16
PAGE???????
???????(polyacrylamide gel)??????? ?(acrylami
de,??Acr)????(crosslinker) N,N-????????(N,N-meth
ylenebisacylamide, ??Bis)????????????????????? ???
?????????,???????????? ???????????
17
????????????

• ??????(Acr)
• ??????????(Bis)
• ???????????(AP)
• ?????????(TEMED)
• ??????????

• ????????????????????(free radicals)???,???????????
  (catalyst-redox systems)????????????????(APTEMED)
  ??????(???TMTED)???

18
???????????
????() ??????(KD)
15 12-43
10 16-68
7.5 36-94
5.0 57-212
SDS-PAGE???(5 Acrylamide)???????http//bbs.bbioo
.com/thread-20726-1-1.html
19
?????
?? ???PH ????
??? ??????? pH6.8Tris-Cl ?,2-5
??? ??????? pH8.8Tris-Cl ?,??????
?????pH8.3 Tris-??????
20
????? ????
ddH2O 0.1SDSlt8 ???gt8
21
????? ????
22
(No Transcript)
23
??

• ??????????????????????(??NC?)?,??????????????????
  ??
• ??????????????????????????,???????/???????????????
  ?

24
??????

• ????Western Blot????????????(Nitrocellulose,NC)???
  ????? (Polyvinylidene Fluoride,
  PVDF)?,?????????DEAE??????????????NC??????,???????
  ??
• ??????????
• http//www.bbioo.com/bio101/2008/21461_2.htm

25
????
26
??????
27
?????(??????)

• ?????
• ??????,?????????????
• ????,?????
• ??????
• ????????????????A???
• ??Western?????

28
??

• ???????????????????????????,?????????,????????????
  ??????
• 5?????BSA(????1h)
• Western Blot ????(??????)
• Tween-20???????????,????????????

29
???????

• ?NC??????????????,??????0.1mL/cm2????????,???????,
  ?????1-2???4????
• ??????,?TBST???3?,??10min?
• ???????????,???????,?????1-2???4????
• ??????,?TBST???3?,??10min?
• ???TBS???2?,??10min?

30
?????????

• ????????(HRP)
• ??????(AP)
• ???????(HRP)

31
?????(??????)

• ????????????
• ?????????????????,??????????????
• ???
• ?????pH???????????????,??????????,????????????
  ??

32
Western Blot????

• ?????????????????????,???????????????????
• Western Blot??????Gel-Pro Analyzer 4
  ???(???????)http//bbs.bbioo.com/thread-44929-1-1.
  html

33
Western Blot??????
34
SDS-PAGE??

• ??????
• ??AP?TEMED?????
• ???????,??????,???????????
• ????,?????????????
• ??????????

• ????
• ?????

35
SDS-PAGE??

• ???????,??????????,????
• ??????,????????,????????
• ????????????????????,????,?????
• ??????????????,???????????????????

• ????????
• ?????????

36
???????

• ???????????????????5-10min
• ?????????????,????????????????????????????
• ????????????
• ??????????,????????????????

• ????????
• ????????
• ????????
• ????????

37
??????????
38
????
39
??????
40
??????
41
Further Readings

• ???Western Blotting??
• http//ask.bbioo.com/special/experiment/WesternBlo
  tting.htm
• Millipore???????
• http//bbs.bbioo.com/thread-45770-1-1.html
• Bio-Rad?western Blotting????
• http//bbs.bbioo.com/thread-30154-1-1.html
• Western blot??????
• http//bbs.bbioo.com/thread-40769-1-1.html

42
????!