銆愮敘(ch菐n)鍝佺法铏�(h脿o)銆�2749380
銆愯嫳鏂囧悕绋便��CAE-Ti-IMAC, 100%
銆愪腑鏂囧悕绋便��鍠垎鏁ｅ浐瀹氬寲瑕拰鑹茶瓬寰悆
銆愮敘(ch菐n)鍝佸収(n猫i)瀹广��250mg, 1g
銆愪繚瀛樻浠躲��瀹ゆ韩鍎�(ch菙)瀛�锛屼繚璩�(zh矛)鏈熺偤浜斿勾銆�
銆愮敘(ch菐n)鍝佹杩般��鏈敘(ch菐n)鍝侀仼鐢ㄤ簬灏�(du矛)纾烽吀鑲界殑閼掑畾鍜屽瘜闆嗐��
銆愯嚜鍌欐潗鏂欍��
•妯ｅ搧锛氳泲鐧介叾瑙ｆ恫
娉ㄦ剰锛氳泲鐧介叾瑙ｆ恫涓笉鑳藉惈鏈夊共鎿剧７閰歌偨瀵岄泦鐨勭墿璩�(zh矛)锛屽EDTA绛夌怠(lu貌)鍚堝姂銆佸惈纾烽吀鏍圭殑绶╂矕楣�锛�
•Loading buffer锛�80%ACN/6%TFA姘存憾娑�
浣滅敤锛氬皣閰惰В娑查吀鍖�锛岄珮婵冨害鐨�TFA-鍙互鎶戝埗閰告�ц偨娈电殑娈樼暀
•Wash buffer 1锛�50%ACN/6%TFA/200 mM NaCl姘存憾娑�
浣滅敤锛氭礂婊岄潪鐗圭暟鎬у惛闄勭殑鑲芥锛岄菇鍙互鎶戝埗闈炵７閰歌偨鐨勬畼鐣�銆�
•Wash buffer 2锛�30%ACN/0.1%TFA姘存憾娑�
浣滅敤锛氭礂婊屾畼鐣欑殑Wash buffer1涓殑楣姐��
•Elution buffer锛�10%姘ㄦ按婧舵恫
銆愭敞鎰忎簨闋�(xi脿ng)銆�
鏈敘(ch菐n)鍝�CAE-Ti-IMAC鏉愭枡鏈灟鍚�Ti4+锛岄鍏堟噳(y墨ng)鍏堝皣Ti4+铻悎鍒版潗鏂欎笂锛岀劧鍚庡啀閫�(j矛n)琛岀７閰歌偨瀵岄泦銆�
銆愬(sh铆)椹�(y脿n)姝ラ銆�
涓�. CAE-Ti-IMAC鏉愭枡鐨�Ti4+铻悎
1. 鎸夌収CAE-Ti-IMAC鏉愭枡锛氱～閰搁垿Ti(SO4)2 = 1:100 (m/m)锛岀ū鍙栨潗鏂欏拰纭吀閳�銆備互绋卞彇300mg鏉愭枡鐐轰緥锛屽皣30g纭吀閳﹀浐楂斿姞鍏�250 mL鐕掔摱涓�锛屽姞鍏�150 mL绱斿噲姘磋秴鑱叉憾瑙ｏ紙娉ㄦ剰锛氱～閰搁垿鍦ㄦ憾瑙ｉ亷绋嬩腑鏈�(hu矛)鏀剧啽锛岃珛(q菒ng)绶╂參鍔犲叆锛夛紝瓒呰伈鍒嗘暎5-10 min锛岀劧鍚庣鍔涙敧鎷�(纾佸姏鏀媽鐨勯�熷害闇�瑕佹帶鍒�锛屽皣鏉愭枡鍕绘伎鍧囧嫽?y脿n)姗�?锛屼笉瀹滈亷蹇垨閬庢參)锛屽婧斁缃�12h銆�
娉ㄦ剰锛� 鍙寜鐓у(sh铆)闅涘埗鍌欐儏娉侀厡鎯呯ū鍙栨潗鏂欏拰纭吀閳�锛屽缓璀�CAE-Ti-IMAC鏉愭枡涓嶄綆浜�250mg銆�
2. 灏囨椹�1涓殑鏉愭枡杞�(zhu菐n)绉昏嚦50 mL闆㈠績绠′腑锛�15000 g闆㈠績3-5 min锛屾涓婃竻銆�
3. 鍔犲叆閬╅噺0.1% TFA姘存憾娑插皣CAE-Ti-IMAC鏉愭枡鍒嗘暎锛岃綁(zhu菐n)绉昏嚦10 mL闆㈠績绠′腑锛屾尟钑╂礂婊屾湭閸靛悎鐨�Ti4+锛�15000 g闆㈠績3-5 min锛屾涓婃竻銆�
4. 閲嶅京(f霉)姝ラ3锛屾礂婊�5-6娆★紝鐩殑鏄洝閲忓幓闄ゆ湭铻悎鐨�Ti4+銆�
5. 鍔犲叆200 mM NaCl/0.1% TFA姘存憾娑诧紝娲楁粚CAE-Ti-IMAC鏉愭枡2娆°��
6. 鍔犲叆0.1% TFA姘存憾娑�锛屾礂婊�2娆�锛屽潎鍕昏綁(zhu菐n)绉昏嚦闆㈠績绠′腑锛屽彲鏍规摎(j霉)瀵�(sh铆)椹�(y脿n)闇�姹傚皣CAE-Ti-IMAC鏉愭枡鍒嗘暎鎴愪笉鍚岀殑婵冨害锛堝100 mg/ml鎴�10 mg/ml锛�锛屼簬4鈩冧繚瀛樸��
7. 妾㈡脯(c猫)CAE-Ti-IMAC鏉愭枡鏄惁鎴愬姛铻悎Ti4+锛氬彇100 uL鏈�鍚庝竴娆℃礂婊岀殑涓婃竻娑�锛屽姞鍏�100 uL 30% H2O2锛堝競鍞洐姘ф按鍘熸恫锛変腑锛岃嫢婧舵恫涓嶈畩鑹诧紝瑾槑铻悎鍚庣殑CAE-Ti-IMAC鏉愭枡宸茬稉(j墨ng)娲楀共鍑�銆傝嫢婧舵恫璁婅壊锛屽墖绻肩簩(x霉)娲楁粚銆傚悓鏅�(sh铆)锛屽彲鍙栧皯閲�CAE-Ti-IMAC鏉愭枡锛屽姞鍏�100 uL 30% H2O2涓�锛岃嫢鏉愭枡鍛堢従(xi脿n)鏄庨’榛冭壊锛岃鏄�Ti4+铻悎鐨�CAE-Ti-IMAC鏉愭枡鍒跺倷鎴愬姛锛屽彲鐢ㄤ簬鍚庣簩(x霉)纾烽吀鑲藉瘜闆嗐��
娉ㄦ剰锛氫笂杩�3-6姝ョ偤CAE-Ti-IMAC鏉愭枡鐨勬礂婊岋紝涓昏鐩殑鏄礂婊屾湭铻悎鐨�Ti4+锛岃珛(q菒ng)鍕跨渷鐣ワ紱鍥犳墍鐢ㄩ洟蹇冩(j墨)涓嶅悓锛岄洟蹇冨姏鍙牴鎿�(j霉)闆㈠績鏁堟灉閬╃暥(d膩ng)瑾�(di脿o)鏁�锛屼繚璀夐洟蹇冨悗CAE-Ti-IMAC鏉愭枡鐒℃嚫娴�锛屼笂娓呮恫婢勬竻銆�
浜�. 婧舵恫娉曞瘜闆嗙７閰稿寲鑲�
1. 铔嬬櫧閰惰В娑茶垏Loading buffer鎸夐珨绌嶆瘮1:1鍧囧嫽娣峰悎锛屽凡铻悎Ti4+鐨�CAE-Ti-IMAC鏉愭枡锛氳泲鐧介叾瑙ｆ恫= 20:1-30:1锛�m/m锛夊潎鍕绘贩鍚�锛岀疆浜庢尟钑╁櫒涓婂婧尟钑�30 min锛岀劧鍚�15000 g闆㈠績3-5 min锛屾涓婃竻銆�
娉ㄦ剰锛�Loading buffer鐢ㄩ噺涓�瀹氳瓒冲锛屼繚璀�TFA绲傛績搴�≥3%锛涘洜CAE-Ti-IMAC鏉愭枡鍦�Ti4+铻悎閬庣▼涓渻(hu矛)鐣ユ湁鎵�鎼嶅け锛屽缓璀版牴鎿�(j霉)瀵�(sh铆)椹�(y脿n)鍏烽珨鎯呮硜锛岃嚜琛屽劒(y艒u)鍖栨潗鏂欒垏妯ｅ搧鐨勪娇鐢ㄦ瘮渚�锛屼互閬�(d谩)鍒版渶鍎�(y艒u)浣跨敤鏁堟灉锛�
2. 鍔犲叆閬╅噺Wash buffer 1锛岄噸鎳告潗鏂�锛屾尟钑�30min锛�15000 g闆㈠績3-5 min锛屾涓婃竻銆�
3. 鍔犲叆閬╅噺Wash buffer 2锛岄噸鎳告潗鏂�锛屾尟钑�30min锛�15000 g闆㈠績3-5 min锛屾涓婃竻銆�
娉ㄦ剰锛氭椹�3鍙噸寰�(f霉)鎿嶄綔锛岀洰鐨勬槸寰瑰簳娓呴櫎NaCl锛屽洜?y脿n)娈乧楣界姝㈤��(j矛n)璩�(zh矛)璀滐紒
4. 鍔犲叆閬╅噺鐨�Elution buffer锛屽啺娴磋秴鑱�5 min锛屾尟钑�15 min锛�20000 g闆㈠績3-5 min锛屾敹闆嗕笂娓呮恫锛堟敞鎰忥細鐩￠噺涓嶈鍚稿埌娌夋穩鐨勬潗鏂欙級銆�
娉ㄦ剰锛氬缓璀伴噸寰�(f霉)姝ラ4锛屽悎骞跺叐娆℃礂鑴恫銆�
5. 灏囧悎骞舵礂鑴恫25000 g闆㈠績3-5 min锛岃綁(zhu菐n)绉讳笂娓呰嚦鏂伴洟蹇冪涓�銆傞洟蹇冩績绺共鐕ュ悗锛�-20鈩冧繚瀛�锛屽緟璩�(zh矛)璀滃垎鏋愩��
娉ㄦ剰锛氭椹�5寰堥噸瑕�锛屼繚璀夋礂鑴恫涓潗鏂欏幓闄ゅ畬鍏紝浠ュ厤鍚庣簩(x霉)鍒嗘瀽鏅�(sh铆)鏉愭枡鍫靛鑹茶瓬鏌�锛�
6. 灏囧噸骞插悗鐨勬ǎ鍝佸京(f霉)婧跺湪涓�瀹氶珨绌嶇殑1%FA姘存憾娑蹭腑锛岃唱(zh矛)璀滃垎鏋�銆�
娉ㄦ剰锛氬瘜闆嗗悗鐨勭７閰歌偨涔熷彲浠ョ敤ZipTip 鎴� StageTip閫�(j矛n)琛岄櫎楣藉悗鍐嶈唱(zh矛)璀滃垎鏋�銆�