灏忛害涓嶅悓閮ㄤ綅铔嬬櫧鍏ㄧ▼鐮旂┒杩囩▼绠�浠?/p>
1.1
灏忛害鍙剁墖铔嬬櫧璐ㄧ粍鎻愬彇
閲囩敤
TCA
锛忎笝閰矇娣�涓�閰氾紡
SDS
鑱斿悎鎶芥彁娉曟潵鎻愬彇灏忛害鍙剁墖铔嬬櫧銆傚弬鐓?/p>
Wang
绛?/p>
[1]
鏂规硶锛?/p>
鍔犱互鏀硅繘銆傚彇鏂伴矞鐨勫皬楹﹀彾鐗?/p>
1g
锛屾恫姘腑杩呴�熺爺纾ㄦ垚缁嗙矇锛屽姞鍏?/p>
10mI
浜庝竴
20
鈩冮鍐风殑鎻愬彇
浠嬭川
[10 TCA(w
锛?/p>
V)
涓欓叜婧舵恫
+0
锛?/p>
2 DTT]
锛?/p>
鍙嶅棰犲�掓贩鍚堬紝
涓�
20
鈩?/p>
娌夋穩
2 h
鎴栬繃澶滐紱
4
鈩?/p>
锛?/p>
16 000
脳
g
绂诲績
10 min
锛屽純涓婃竻锛涙矇娣�鐢ㄥ惈
100mmol
锛?/p>
I
閱嬮吀閾电殑
80
鐢查唶婧舵恫娲?/p>
1
娆★紝
80
涓欓叜娲?/p>
1
娆★紝
閫氶姗卞唴瀹ゆ俯骞茬嚗
10 min
锛?/p>
鍔犲叆
10mI
閰氾紡
SDS
鎶芥彁澶?/p>
[-pH 8
锛?/p>
0 Tris
楗卞拰閰氫笌
SDS
缂撳啿娑?/p>
(30
钄楃硸銆?/p>
2 SDS
銆?/p>
5 J3
涓�宸熀涔欓唶銆?/p>
0
锛?/p>
1mol
锛?/p>
[ pH 8
锛?/p>
0 Tris
鈥?/p>
Hc1)1
锛?/p>
1}
鏄嗗悎
]
鎸崱娣峰寑娓╄偛
5 min
锛?/p>
4
鈩?/p>
锛?/p>
16 000
脳
g
绂诲績
10 min
锛?/p>
杞Щ涓婂眰閰氱浉鑷充竴骞插噣鏂扮涓紝
鍔犲叆
5
鍊嶄綋绉殑鍐风殑鍚?/p>
100mmol
锛?/p>
I
閱嬮吀閾电殑鐢查唶婧舵恫锛屼竴
20
鈩?/p>
娌夋穩
4 h
鎴栬繃澶滐紱
4
鈩?/p>
锛?/p>
16 000
脳
g
绂诲績
lOmin
锛屽純涓婃竻娑诧紝
娌夋穩鐢ㄧ敳閱囨礂
1
娆★紝
80
涓欓叜娲?/p>
1
娆★紝閫氶姗卞唴鑷劧椋庡共锛岋綖
80
鈩?/p>
鍐扮淇濆瓨澶囩敤銆?/p>
1.2
灏忛害鑳氫钩铔嬬櫧鐨勬彁鍙?/p>
璧?/p>
[2]
閫夌敤
TCA-
涓欓叜娉曪紝鎶界鏃堕�夊彇鍚屼竴澶╂娊绌楁寕鐗屾爣璁帮紝鎶界鍚?/p>
10 d
鍙栨寕鐗屾爣璁扮殑
5
涓?/p>
绌楋紝鍙栫涓笂閮ㄧ亴娴嗕竴鑷寸殑绫界矑锛屽墺鍘婚澹崇疆浜庡喕瀛樼涓?/p>
-80
鈩冧繚瀛樸�傚彇鍐诲瓨绫界矑
20
绮掞紝鐢?/p>
鐏弻棰勫喎鐨勫壀鍒�鍜岄晩瀛愬湪棰勫喎鐨勭爺閽典腑杩呴�熷墺鍘荤鐨紝
鍒囧幓鑳氾紝
鍔?/p>
0.2% PVP
杩呴�熺爺纾ㄨ嚦绯婄姸锛?/p>
鎮诞浜庡惈
0.07% DTT
鐨勯鍐?/p>
10%TCA-
涓欓叜锛?/p>
-20
鈩冭繃澶溿�?/p>
娆℃棩
4
鈩?/p>
35 000 r
路
min-1
绂诲績
15 min
锛?/p>
寮冧笂娓咃紝
灏嗘矇娣�閲嶆偓浜?/p>
80%
涓欓叜锛屼簬
-20
鈩冩俯娴?/p>
l h
锛?/p>
4
鈩?/p>
16 000 r
路
min-1
绂诲績
15 min
銆傛矇娣�
閲嶆偓浜?/p>
100%
涓欓叜锛屼簬
-20
鈩冩俯娴?/p>
l h
锛?/p>
4
鈩?/p>
16 000 r
路
min-1
绂诲績
15 min
銆傞噸澶嶆姝ラ
2~4
娆★紝
鍊掑嚭涓婃竻娑诧紝绂诲績绠＄疆浜庡啺娴翠腑鎶界湡绌?/p>
15 min
鑷充笝閰畬鍏ㄦ尌鍙戯紝寰楀埌鐨勮泲鐧戒负绮楄泲鐧姐�?/p>
1.
鍙屽悜鐢垫吵鎶�鏈?/p>
1.1
灏忛害鍙剁墖铔嬬櫧璐ㄧ粍鍙屽悜鐢垫吵鎶�鏈?/p>
鍙傜収鏉?/p>
[3]
閲囩敤
SDS
鈥?/p>
PAGE
鍜屽弻鍚戝嚌鑳剁數娉?/p>
30 mg
铔嬬櫧绮夊姞鍏?/p>
300 FI
姘村寲涓婃牱缂撳啿娑?/p>
(8mol
锛?/p>
I
灏跨礌銆?/p>
4 CHAPS
銆?/p>
65 mmol
锛?/p>
I DTT
銆?/p>
0
锛?/p>
2V
锛?/p>
V BioIyte)
锛?/p>
37
鈩冩按娴?/p>
30 min
锛?/p>
6
鈩?/p>
锛?/p>
40 000
脳
g
绂诲績
30 rain
锛?/p>
鍙栦笂娓呮恫锛?/p>
Bradford
娉曟祴瀹氳泲鐧芥祿搴?/p>
銆?/p>
SDS
鈥?/p>
PAGE
鐢垫吵涓婃牱閲忎负
100 mg
锛岃泲
鐧芥牱鍝佷笌
2
脳鐢垫吵涓婃牱缂撳啿娑茬瓑浣撶Н娣峰悎锛?/p>
鐩存帴鍦?/p>
12
鐨勮仛涓欑儻閰拌兒鍑濊兌涓數娉筹紝
鑳朵綋鑰冩煋鍙?/p>
锛?/p>
鐢ㄦ櫘閫氬嚌鑳舵垚鍍忕郴缁?/p>
(Bio
鈥?/p>
Rad
锛?/p>
缇庡浗
)
閲囬泦鍥惧儚銆?/p>
鍙屽悜鐢垫吵閲囩敤
BioRad
鍏徃鐢垫吵绯荤粺
(Protean
IEF cell
绛夌數鑱氱劍绯荤粺銆?/p>
PR()TEAN 11 xi Cell
鍨傜洿鐢垫吵绯荤粺銆?/p>
Mini pro
鈥?/p>
tean
鈪?/p>
cell
鍨傜洿鐢垫吵绯荤粺
)
锛?/p>
铔嬬櫧涓婃牱閲忎负锛?/p>
7 cm
鑳舵潯
200 Fg
銆?/p>
17 cm
鑳舵潯
5OO Fg
銆傜瓑鐢佃仛鐒﹀拰鍚戠浜屽悜
SDS
鈥?/p>
PAGE
鐨勮浆绉?/p>
鍙傜収
Bio
鈥?/p>
Rad
鎿嶄綔鎸囧崡杩涜銆?/p>
IPG
鑳舵潯涓?/p>
7
銆?/p>
17 cm(pH
鍊?/p>
3
锝?/p>
10
銆?/p>
4
锝?/p>
7
锛?/p>
Bio
鈥?/p>
Rad)
锛岀浜屽悜鑱?/p>
涓欑儻閰拌兒鍑濊兌鐢垫吵鑳舵祿搴︿负
12
銆?/p>
鏌撹壊閲囩敤鑳朵綋鑰冮┈鏂寒钃濇煋鑹叉柟娉?/p>
l1
銆?/p>
UMAX Power
鈥?/p>
I
锛?/p>
ook
2100XI
鍨嬪厜瀵嗗害鎵弿浠?/p>
(Bio
鈥?/p>
Rad
锛岀編鍥?/p>
)
锛屽垎杈ㄧ巼璁句负
300dpi
閲囬泦鍥惧儚銆傛瘡绉嶈泲鐧芥彁鍙栨柟娉?/p>
鐨勫弻鍚戠數娉宠繘琛?/p>
3
娆￠噸澶嶃�?/p>
PDQuest 7
锛?/p>
4
杞欢
(BioRad
锛岀編鍥?/p>
)
瀵瑰浘鍍忚繘琛屽垎鏋愩�?/p>
2.2
灏忛害鑳氫钩铔嬬櫧鐨勫弻鍚戠數娉?/p>
瀵瑰皬楹﹁儦涔抽噰鐢ㄤ互涓嬫妧鏈繘琛岀數娉冲璧?/p>
[4].
鏍峰搧琚姩涓婃牱鍙婄瓑鐢佃仛鐒?/p>
,
閫夌敤
17 cm IPG
鑳舵潯锛?/p>
pH 4~7
锛?/p>
銆?/p>
IPG
骞茶兌鏉¤兌闈㈡湞涓嬫斁鍏ユ按鍖栨恫鐨勬按浠ｇ洏涓紝瑕嗙洊涓�灞傜熆鐗╂补浠ラ槻姝㈢瓑鐢佃仛
鐒︽椂灏跨礌鏋愬嚭锛岃泲鐧芥哀鍖栧強浜х敓鍐峰嚌姘达紝琚姩姘村寲
14 h
鍚庯紝缃簬
Bio-Rad
绛夌數鑱氱劍浠腑锛岀瓑
鐢佃仛鐒﹀湪
20
鈩冩潯浠朵笅鑷姩杩涜锛屾瘡鑳舵潯闄愭祦
50
渭
A
銆傜瓑鐢佃仛鐒﹀弬鏁板涓嬶細鈶?/p>
500 V 4 h
闄?/p>
鐩愶紱鈶?/p>
1 000 V 1 h
绾挎�т笂鍗囷紙
Gra
锛?/p>
锛涒憿
8 000 V 2.5 h
绾挎�т笂鍗囷紙
Gra
锛?/p>
锛涒懀
8 000 V 0.5 h
鑱?/p>
鐒︺�?/p>
鑳舵潯鐨勫钩琛?/p>
,
灏嗙瓑鐢佃仛鑳跺悗鐨勮兌鏉¤兌闈㈠悜涓婄疆浜?/p>
5 mL
骞宠 缂撳啿娑测厾
锛?/p>
50 mol
路
L-1 Tris-HCL
锛?/p>
pH 8.8
锛?/p>
6 mol
路
L-1urea
锛?/p>
30%
鐢樻补锛?/p>
2% SDS
锛?/p>
0.002%
婧撮厷钃濓紝
1%DTT
锛変腑骞宠 鎽囧簥闇囪崱
15 min
锛岀劧鍚庡啀缃簬
5 mL
骞宠 缂撳啿娑测叀锛?/p>
50 mmol
路
L-1 Tris-HCL
锛?/p>
pH 8.8
锛?/p>
6 mol
路
L-urea
锛?/p>
30%
鐢樻补锛?/p>
2% SDS
锛?/p>
0.002%
婧撮厷钃濓紝
2.5%
纰樹箼閰拌兒锛変腑骞宠
15 min
锛屽彇鍑哄悗鏀惧湪婀挎鼎鐨勫畾鎬ф护
绾镐笂锛屼互鍚稿幓琛ㄩ潰澶氫綑鐨勬恫浣撱�?/p>
SDS-PAGE,
閲囩敤
Bio-Rad
鍨傜洿鐢垫吵绯荤粺锛?/p>
鑳剁殑娴撳害涓?/p>
12%
锛?