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2. 2mol/L NaCL 婧舵恫 50mL
3. 1mol/L NaOH婧舵恫 50mL
4. 0.2mol/L NiSO4婧舵恫 50mL
5. 骞宠 绶╂矕娑诧細50mmol/L Tris-HCL锛�500mmol/L NaCL锛宲H7.0 500mL
6. Ni2锛� Chelating Sepharose Fast Flow 5锛�10 mL
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10 20%涔欓唶婧舵恫50ml
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1. 妯ｅ搧鐨勫埗鍌�
绱�(x矛)鑳炵殑鍩归(y菐ng)鍙婄啋鍏夎泲鐧借〃閬�(d谩)鐪嬪(sh铆)椹�(y脿n)鍗�锛岀窗(x矛)鑳炵殑鐮寸鍙婅泲鐧界殑鏀堕泦濡備笅锛氭敹闆嗗湪25 鈩冪敤绱�(x矛)鑳炲煿椁�(y菐ng)娑�锛�8000r/min锛岄洟蹇�5min锛屽幓涓婃竻娑�锛岃弻楂旂敤骞宠 绶╂矕娑叉礂婊屼竴娆�锛岄洟蹇冩敹闆嗚弻楂�锛岀敤涓夊垎涔嬩竴锛堢窗(x矛)鑳炲煿椁�(y菐ng)娑诧級楂旂⿳鐨勫钩琛＄珐娌栨恫鍏呭垎鎳告诞锛屽啺娴翠笅閫�(j矛n)琛岄珮澹撶牬鑿岃檿鐞�銆傜劧鍚�8000r/min銆傞洟蹇�30min锛屽彇涓婃竻娑�銆傛斁鍐扮鍌欑敤銆�
2. 瑕拰灞ゆ瀽鏌辩殑瀹夎
鎶婂堡鏋愭煴鍥哄畾鍦ㄩ惖鏀灦涓�锛屼笂绔殑鏌遍牠鎿颁笅锛屾煴涓嬬鍑哄彛鐢ㄥぞ瀛愬皝闁�銆傚姞鍏ュ皯閲忕殑鐒￠洟瀛愭按锛屾帓鍘讳笅绔殑绌烘埃娉�銆傚彇鍑�20%涔欓唶娴告场鐨勮灟鍚堝嚌鑶�5锛�10mL鍒扮噿鏉腑锛屽姞鍏ュ皯閲忕殑鐒￠洟瀛愭按鍒舵垚绯婄媭锛屾部钁楄布绶婃煴鍏�(n猫i)澹佺殑鐜荤拑妫掓妸绯婄媭鍑濊啝鍊掗��(j矛n)鏌卞収(n猫i)锛屾墦闁嬩笅绔殑鎺掓按鍙�锛岃畵瑕拰鍑濊啝鍔戦毃姘存祦鑷劧娌変笅銆傝Κ鍜屽堡鏋愬姂鐐�5鈥�10mL銆傜劧鍚�锛屽皣涓婄鐨勬煴闋摪绶婏紝骞跺皣闋傜鍜屼笅绔敤杌熺閫ｆ帴灏侀枆鍌欑敤銆�
3. 灞ゆ瀽绯荤当(t菕ng)鐨勫畨瑁濄�佽(di脿o)瑭�
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锛�2锛夌郴绲�(t菕ng)鐨勯�ｆ帴锛氬皣锠曞嫊娉电殑鍑烘按绠¤垏灞ゆ瀽鏌变笂绔鐩搁�ｏ紝灞ゆ瀽鏌辩殑涓嬬绠¤垏绱妾㈡脯鍎�鐨勯��(j矛n)妯ｇ閫ｆ帴锛屽皣绱妾㈡脯鍎�鐨刼ut绔垏鑷嫊鏀堕泦鍣ㄧ浉閫ｃ��
锛�3锛夌传澶栨敹闆嗗櫒鐨勮(di脿o)瑭︼細鎵撻枊绱鑳屽悗闁嬮棞(gu膩n)锛岃(di脿o)绡�(ji茅)闈堟晱搴︽棆閳曪紙ABS锛峈ange锛夛紝瑾�(di脿o)绡�(ji茅)瑾�(di脿o)闆舵棆閳�銆�
锛�4锛夎嚜鍕曟敹闆嗗櫒鐨勮ō(sh猫)缃細鎵撻枊闆绘簮闁嬮棞(gu膩n)锛屾寜鈥滃京(f霉)浣嶁�濋嵉锛岀⒑瀹氬嚭娑茬鐨勪綅缃紝鎸夆�滄墜鍕曗�濇寜閳�锛岄��(j矛n)琛岃ō(sh猫)缃紝濡傞伕鎿�120seconds涓�绠￠��(j矛n)琛屾敹闆�锛屾寜鈥滆嚜鍕曗�濆嵆闁嬪瑷�(j矛)鏅傛敹闆�銆�
锛�5锛変紞璞壊璀滃伐浣滅珯鐨勪娇鐢ㄦ柟娉曪細榛�(di菐n)鎿婃闈㈢殑鈥滀紞璞壊璀滃伐浣滅珯鈥滃湒妯�(bi膩o)鎵撻枊绋嬪簭锛岄粸(di菐n)鎿婂乏涓婅鐨勫湒妯�(bi膩o)锛岄伕鎿嘇鎴朆閫氶亾锛岄粸(di菐n)鎿娾�溾柌鈥濆嵆闁嬪閲囨ǎ锛岄噰妯ｇ祼(ji茅)鏉熷悗榛�(di菐n)鎿娾�溾枲鈥濆嵆绲�(ji茅)鏉熼噰妯�锛岀劧鍚庤級鍏鎴朆閫氶亾璀滃湒锛屼繚瀛樼祼(ji茅)鏋�锛屽苟鎵撳嵃绲�(ji茅)鏋溿��
4. 灞ゆ瀽鏌辩殑浣跨敤鍓嶈檿鐞嗭細
锛�1锛夌敤5X鏌卞簥楂旂⿳鍘婚洟瀛愭按娓呮礂涔欓唶灏佸瓨鐨凬i 2锛�-Chelating Sepharose Fast Flow瑕拰灞ゆ瀽鏌憋紝鍘婚櫎涔欓唶
锛�2锛夌敤2X鏌卞簥楂旂⿳鐨�0.2M NiSO4閬庢煴锛屾敞鎰忚瀵熺従(xi脿n)璞�
锛�3锛� 鐢�10X鏌卞簥楂旂⿳鐨勫幓闆㈠瓙姘存竻娲�锛岀敤5X鏌卞簥楂旂⿳骞宠 绶╂矕娑插钩琛℃煴瀛�
5. 涓婃ǎ锛�
鍏堝緸20 mL瑁傝В涓婃竻娑蹭腑鍙栧嚭50 渭L鐢ㄤ簬闆绘吵锛屼綔鐐鸿Κ鍜屽堡鏋愬垎闆㈠墠涓婃竻娑蹭腑鐨勭附铔嬬櫧鍗�(q奴)甯跺皪鐓у湒璀溿�傜劧鍚庝互姣忓垎閻�2mL鐨勬祦閫熶笂灞ゆ瀽鏌憋紝鍒嗛儴鏀堕泦娴佸嚭娑诧紝姣忕3 mL锛堣閷勭殑绱鍚告敹宄扮偤绌挎祦宄帮紝鍙杬ui楂樼殑涓�绠℃ǎ鍝佹恫鐢ㄤ綔闆绘吵锛�銆傚钩琛＄珐娌栨恫閬庡堡鏋愭煴锛岀洿鍒扮传澶栧惛鏀跺嘲涓嶅啀涓嬮檷锛堥仈(d谩)鍒板钩鑷烘湡鐐烘锛�銆�
6. 娲楁粚锛�
鐢ㄥ惈50 mmol/L鍜攽鐨勬礂婊岀珐娌栨恫25 mL鍐艰劔锛屽垎閮ㄦ敹闆嗘礂鑴恫锛屾瘡绠�5 mL锛屽彇绱鍚告敹zui楂樼殑涓�绠＄敤浜庨浕娉炽��
7. 娲楄劔锛�
鐢ㄥ惈300mmol/L鍜攽鐨勬礂鑴珐娌栨恫10 mL娲楄劔锛岃嚜鍕曟敹闆嗘礂鑴恫銆傛瘡绠℃敹5 mL锛岀暥(d膩ng)绱鍚告敹閬�(d谩)zui楂樺嘲鏅傚彇妯ｇ敤浜庨浕娉冲強Western blotting瀵�(sh铆)椹�(y脿n)銆�
8. 灞ゆ瀽鏌辩殑鍚庤檿鐞嗗強灏佸瓨锛�
锛�1锛夌敤2X鍘婚洟瀛愭按娓呮礂鏌卞瓙
锛�2锛夌敤10X0.5M NaCl锛�50mM EDTA (pH 8.0) 娲楀幓绲�(ji茅)鍚堢殑閹抽洟瀛�
锛�3锛夌敤10X鍘婚洟瀛愭按娓呮礂鏌卞瓙
锛�4锛夌敤10X1M NaOH娲楁煴锛屽幓娈樼暀铔嬬櫧
锛�5锛夌敤澶х磩10X鍘婚洟瀛愭按娓呮礂鏌�锛屽幓闄aOH锛岀洿鍒皃H鍊间綆浜�
锛�6锛夌敤澶х磩2X鏌卞簥楂旂⿳涔欓唶閬庢煴锛屾媶闄ゆ煴瀛愶紝淇濆瓨鏌辨枡浜�20锛呬箼閱囦腑
9. 12%SDS-鑱氫笝鐑叞鑳哄嚌鑶犻浕娉虫娓細
鍒嗗垾鍙�50渭L绌挎祦宄版祦鍑烘恫銆�50mmol/L鍜攽绶╂矕娑叉礂婊岀殑娴佸嚭娑插拰300mmol/L鍜攽绶╂矕娑叉礂鑴殑娴佸嚭娑层�傚鍔犱竴鍊嬩笂鏌卞墠鐨勬ǎ鍝佹恫鍜岃泲鐧組arker浣滄瘮杓冦�傞��(j矛n)琛�12% SDS-鑱氫笝鐑叞鑳哄嚌鑶犻浕娉虫娓Κ鍜屽堡鏋愬垎闆㈢磾鍖栫殑绲�(ji茅)鏋�銆�
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