绮惧搧鏂囨。
绮惧搧鏂囨。
澶ц偁鏉嗚弻琛ㄨ揪閲嶇粍铔嬬櫧鐨勮秴澹扮牬纰庡強绾寲
涓�
鍙憾鎬ц泲鐧界殑绾寲
锛堜竴锛夎弻浣撶殑鐮寸
1.
浠櫒涓庢潗鏂欙細
-80
鈩冨啺绠憋紱
瓒呭０娉㈢粏鑳炵牬纰庝华锛?/p>
50mM PBS
鎴?/p>
50mM Tris-HCl pH
7.5
锛?/p>
50ml
绂诲績绠★紱鍐峰喕楂橀�熺蹇冩満
2.
鏂规硶
2.1
鍙嶅鍐昏瀺
2.1.1
鏀堕泦鑿屾恫
500ml
锛岀瓑鍒?/p>
10
浠斤紝
4000 r/min
4
鈩冪蹇?/p>
15min
锛屽純涓婃竻銆?/p>
2.1.2
鑿屼綋娌夋穩涓姞鍏ョ浉鍚岃弻娑蹭綋绉殑
50mM PBS
鎴?/p>
50mM Tris-HCl
锛堥�夋嫨浣胯泲
鐧界ǔ瀹氱殑缂撳啿娑插拰
pH
锛夐噸鎮礂娑や竴娆°�?/p>
2.1.3
鐒跺悗鎸夊師鑿屾恫浣撶Н鐨?/p>
1/4
鍔犲叆缂撳啿娑查噸鎮弻浣擄紝
骞跺姞鍏ヨ泲鐧介叾鎶戝埗鍓?/p>
PMSF
鍜?/p>
EDTA(
甯?/p>
His
鏍囩涓嶅姞
)
锛?/p>
PMSF
缁堟祿搴︿负
100渭g/ml,
EDTA
鐨勭粓娴撳害涓?/p>
銆傚彇
20渭l
閲嶆偓鑿屾恫杩涜鐢垫吵锛?/p>
妫�娴嬭泲鐧借〃杈剧殑鎯呭喌
锛堟槸鍚﹁〃杈撅紝
鏄彲婧舵�ц〃杈捐繕鏄寘
娑典綋琛ㄨ揪锛?/p>
銆?/p>
2.1.4
灏嗚弻娑诧紙缁忔娴嬫湁琛ㄨ揪锛夊湪
-80
搴﹀啺鍐伙紝瀹ゆ俯铻嶈В锛屽弽澶嶅嚑娆★紙鍙嶅鍐昏瀺涓?/p>
娆★級
锛岀敱浜庣粏鑳炲唴鍐扮矑褰㈡垚鍜屽墿浣欑粏鑳炴恫鐨勭洂娴撳害澧為珮寮曡捣婧惰儉锛屼娇缁嗚優缁撴瀯鐮?/p>
纰庛�?/p>
2.2
瓒呭０娉㈠鐞?/p>
(
瀵硅秴澹版尝鍙婄儹鏁忔劅鐨勮泲鐧芥厧鐢?/p>
)
2.2.1
灏嗗弽澶嶅喕铻嶇殑鑿屾恫
锛堝繀瑕佹椂鍙姞鍏?/p>
1mg/ml
婧惰弻閰讹紝
缂撳啿娑?/p>
pH
锛?/p>
8.0
锛?/p>
鍔犲叆
鍚庨渶闈欑疆
20min
锛?/p>
锛岃繘琛岃秴澹扮牬纰庯紝瓒呭０鏉′欢锛?/p>
400W
锛屽伐浣?/p>
5
绉掞紝闂撮殧
5
绉掞紝閲?/p>
澶嶄竴瀹氭鏁帮紝
锛堟牴鎹垜浠殑浠櫒鎵惧嚭涓�涓瘮杈冨ソ鐨勫伐浣滄潯浠讹級
銆?/p>
鐩磋嚦鑿屼綋婧舵恫鍙樻竻
婢堜负姝紝澶х害鑺辫垂鏃堕棿
銆?/p>
2.2.2
鍙栧皯閲忕粡瓒呭０鐮寸鍚庣殑鑿屾恫锛?/p>
10000rpm
绂诲績
10
鍒嗛挓锛屽垎鍒涓婃竻鍜屾矇娣�
杩涜妫�娴嬶紝
骞剁敤鍏ㄨ弻浣滀负闃虫�у鐓э紝
妫�娴嬭弻浣撶牬纰庣▼搴﹀強鐩爣鏉″甫鍗犳�昏泲鐧界殑鍚?/p>
閲忋�?/p>
娉ㄦ剰浜嬮」锛?/p>
锛?/p>
1
锛?/p>
瓒呭０鐮寸鍏蜂綋鏉′欢鍙牴鎹疄楠屾儏鍐佃�屽畾锛岃鎺屾彙濂藉姛鐜囧拰姣忔瓒呭０鏃堕棿锛?/p>
闄嶄綆铔嬬櫧琚檷瑙ｇ殑鍙兘銆?/p>
锛?/p>
2
锛?/p>
鍔熺巼澶ф椂锛屾瘡娆¤秴澹版椂闂村彲缂╃煭锛屼笉鑳借娓╁害鍗囬珮锛屽簲淇濇寔鍦?/p>
4
搴﹀乏鍙筹紝
瓒呭０鏃朵繚鎸佸啺娴淬�?/p>
锛?/p>
3
锛夎弻浣撶牬纰庡悗鎬昏泲鐧芥祿搴︾殑娴嬪畾鍙敤
Bradford
娉曟垨鑰呯传澶栧惛鏀舵硶銆?/p>
锛?/p>
4
锛夊彲閫氳繃
SDS-PAGE
鐢垫吵瑙傚療鑿屼綋鐮寸绋嬪害鍙婄洰鏍囨潯甯﹀崰鎬昏泲鐧界殑鍚噺銆?/p>
浜?/p>
鍖呮兜浣撹泲鐧界殑绾寲
1
鑿屼綋鐨勭牬纰?/p>
锛堝姞婧惰弻閰跺鐞嗗寘娑典綋鏁堟灉鍙兘涓嶅ソ锛屽寘娑典綋涓�绘槸鏈夋畫鐣欑殑婧?/p>
鑿岄叾锛屼綘鐪嬬湅鏈夋病鏈変笉鍔犳憾鑿岄叾鐨勶紝杩欎釜鍏堜繚鐣欏ソ浜嗭級
1.1
浠櫒涓庢潗鏂欙細瓒呭０娉㈢粏鑳炵牬纰庝华锛?/p>
20mM PBS
鎴?/p>
20mM Tris-HCl pH 7.5
锛涜
瑙ｆ恫
buffer A
锛涙憾鑿岄叾
10mg/ml
锛?/p>
50ml
锛?/p>
15ml
绂诲績绠★紱鍐峰喕绂诲績鏈?/p>
1.2
鏂规硶
(1)
鏀堕泦鑿屾恫
500ml
锛岀瓑鍒?/p>
10
浠斤紝
4000 r/min 4
鈩冪蹇?/p>
15min
锛屽純涓婃竻銆?/p>