銆愮敘(ch菐n)鍝佺法铏�(h脿o)銆�2749381
銆愯嫳鏂囧悕绋便��SPE-Ti-IMAC, 100%
銆愪腑鏂囧悕绋便��鍥虹浉钀冨彇鍥哄畾鍖栬Κ鍜岃壊璀滃井鐞�
銆愮敘(ch菐n)鍝佸収(n猫i)瀹广��250mg, 2g
銆愮敘(ch菐n)鍝佸収(n猫i)瀹广��瀹ゆ韩鍎�(ch菙)瀛橈紝淇濆瓨鏈熺偤浜斿勾銆�
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�銆�
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╂矕楣斤紒
•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: 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閰歌偨鐨勬畼鐣欍��
•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�銆�
•Elution buffer锛�10%姘ㄦ按婧舵恫
•鑰楁潗锛�200 μL妲嶉牠銆�600 ul闆㈠績绠�銆�1.5 ml闆㈠績绠°�佺洿寰戠偤1 mm鐨勭鏉�
•Tip鏌辨簴(zh菙n)鍌�锛�
1) 灏�1 mm鐨勭鏉垮～鍏�200 μL妲嶉牠涓�锛屽鍦�1a-b鎵�绀猴紝鐩″彲鑳藉～鐨勯潬杩戞闋殑搴曠銆�
鍦�1
2) 濡傚湒2鎵�绀�锛屽皣600 μL闆㈠績绠″壀闁�(k膩i)锛屽湪闆㈠績绠¤搵涓枔鎵撳瓟锛屼娇200 μL妲嶉牠鍙互鍗″湪鍦�1c鎵�绀轰綅缃紱
鍦�2
3) 濡傚湒1d鎵�绀�锛�1 mL闆㈠績绠″壀鎺夎搵瀛愪綔鐐哄绠�锛岀劧鍚庡湪200 μL鐨勬闋腑鍔犲叆閬╅噺宸茶灟鍚�Ti4+鐨�SPE-Ti-IMAC鏉愭枡锛�Tip鏌卞埗浣滃畬鎴�锛屽鍦�3鎵�绀�銆�
鍦�3
銆愭敞鎰忎簨闋�(xi脿ng)銆�
鏈敘(ch菐n)鍝�SPE-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�
200 ul妲嶉牠涓�鑸缓璀版渶澶氳10 mg SPE-Ti-IMAC鏉愭枡锛屽鏋滄ǎ鍝佽捣濮嬮噺杓冨ぇ锛岃珛(q菒ng)鎸夋瘮渚嬪鍔�SPE-Ti-IMAC鏉愭枡浣跨敤閲忋�傜暥(d膩ng)鏉愭枡浣跨敤閲忕偤100 mg浠ヤ笅锛屽彲浠ヤ娇鐢ㄦ煴绠￠珨绌嶇偤1 ml鐨�SPE鏌辩鍙婂叾灏�(du矛)鎳�(y墨ng)绡╂澘锛涚暥(d膩ng)鏉愭枡鐢ㄩ噺鐐�100-200 mg鏅�(sh铆)锛屽彲浣跨敤鏌辩楂旂⿳鐐�3 ml宸﹀彸鐨�SPE鏌辩鍙婂叾灏�(du矛)鎳�(y墨ng)绡╂澘銆�
銆愬(sh铆)椹�(y脿n)姝ラ銆�
涓�銆� SPE-Ti-IMAC鏉愭枡鐨�Ti4+铻悎
1. 鎸夌収SPE-Ti-IMAC鏉愭枡:纭吀閳�Ti(SO4)2 = 1:50 (m/m)锛岀ū鍙栨潗鏂欏拰纭吀閳�銆備互绋卞彇500 mg鏉愭枡鐐轰緥锛屽皣25 g纭吀閳﹀浐楂斿姞鍏�50 mL骞冲簳闆㈠績绠′腑锛屽姞鍏ョ磾鍑堟按鑷�40 ml锛岃秴鑱叉憾瑙� (娉ㄦ剰锛氱～閰搁垿鍦ㄦ憾瑙ｉ亷(gu貌)绋嬩腑鏈�(hu矛)鏀剧啽锛岃珛(q菒ng)绶╂參鍔犲叆)銆傚悜纭吀閳︽憾娑蹭腑鍔犲叆SPE-Ti-IMAC鏉愭枡锛屾贩鍕�锛岀疆浜�Rolling Incubator涓婃悥鍕�锛屽婧亷(gu貌)澶�锛岃珛(q菒ng)鍕跨鍔涙敧鎷�銆�
娉ㄦ剰锛氬彲鎸夌収瀵�(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�SPE-Ti-IMAC鏉愭枡涓嶈浣庝簬500 mg銆�
2. 娆℃棩锛屽皣姝ラ1涓殑SPE-Ti-IMAC鏉愭枡闈滅疆鑷冲叾瀹屽叏娌夊埌闆㈠績绠″簳閮紝妫勪笂娓�銆�
3. 鍔犲叆閬╅噺0.1% TFA姘存憾娑插皣SPE-Ti-IMAC鏉愭枡鍒嗘暎锛屾贩鍕伙紝闈滅疆鑷冲叾瀹屽叏娌夊埌闆㈠績绠″簳閮�锛屾涓婃竻銆�
4. 閲嶅京(f霉)姝ラ3锛屾礂5-6娆″乏鍙�锛岀洰鐨勬槸鐩￠噺鍘婚櫎鏈灟鍚堢殑Ti4+銆�
5. 鍔犲叆200 mM NaCl/0.1% TFA姘存憾娑叉礂婊�SPE-Ti-IMAC鏉愭枡2娆�銆�
6. 鍔犲叆0.1% TFA姘存憾娑叉礂婊�2娆★紝鍧囧嫽杞�(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SPE-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�銆�
7. 妾㈡脯(c猫)SPE-Ti-IMAC鏉愭枡鏄惁鎴愬姛铻悎Ti4+锛氬彇100 uL鏈�鍚庝竴娆℃礂婊岀殑涓婃竻娑诧紝鍔犲叆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�锛岃(shu艒)鏄庤灟鍚堝悗鐨�SPE-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�SPE-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(shu艒)鏄�Ti4+铻悎鐨�SPE-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�銆�
娉ㄦ剰锛氫笂杩�3-6姝ラ鐐�SPE-Ti-IMAC鏉愭枡鐨勬礂婊�锛屼富瑕佺洰鐨勬槸娲楁粚鏈灟鍚堢殑Ti4+锛岃珛(q菒ng)鍕跨渷鐣�銆�
浜�. Tip (SPE)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�SPE-Ti-IMAC锛氳泲鐧介叾瑙ｆ恫= 20:1-30:1锛�m/m锛夊潎鍕绘贩鍚�銆備互涓嬩互250 ug铔嬬櫧閰惰В娑�锛�SPE-Ti-IMAC鏉愭枡锛氳泲鐧介叾瑙ｆ恫=20:1鐐轰緥銆�
娉ㄦ剰锛�Loading buffer涓�瀹氳瓒抽噺锛屼繚璀�TFA绲傛績搴�≥3%锛涘洜SPE-Ti-IMAC鏉愭枡鍦�Ti4+铻悎閬�(gu貌)绋嬩腑鏈�(hu矛)鐣ユ湁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. 鍚�5 mgTi4+铻悎鐨�SPE-Ti-IMAC Tip鏌变腑鍔犲叆200 μL 0.1%TFA锛屾竻娲楁潗鏂�銆�
3. 250 μL铔嬬櫧閰惰В娑诧紙1 μg/μL锛夎垏Loading buffer鎸夐珨绌嶆瘮1:1鍏呭垎娣峰悎锛屽姞鍏ュ埌Tip鏌变腑閫�(j矛n)琛屽瘜闆�锛岀附鏅�(sh铆)闁撴帶鍒跺湪20 min宸﹀彸銆�
4. 鍔犲叆200 μL Wash buffer 1娲楁粚锛岄櫎鍘婚潪鐗圭暟鎬у惛闄�锛岄噸寰�(f霉)1娆★紝绺芥檪(sh铆)闁撴帶鍒跺湪10 min宸﹀彸銆�
5. 鍔犲叆200 μL Wash buffer 2娲楁粚1娆�锛岄櫎鍘讳笂杩版椹熶腑娈樼暀鐨勯菇锛屾檪(sh铆)闁撴帶鍒跺湪5 min宸﹀彸銆�
6. 鍔犲叆200 μL Elution buffer娲楄劔2娆�锛屽悎骞舵礂鑴恫锛屽嵆瀵岄泦濂界殑纾烽吀鑲�锛岀附鏅�(sh铆)闁撴帶鍒跺湪15 min宸﹀彸銆�
7. 娲楄劔鐨勬ǎ鍝佸湪鍑嶅共姗�(j墨)涓噸骞�锛屼簬-20鈩冧繚瀛�锛岃唱(zh矛)璀滃垎鏋愭檪(sh铆)闇�鐢�1% FA寰�(f霉)婧�銆�
娉ㄦ剰锛�Tip娉曞瘜闆嗙７閰稿寲鑲戒竴鑸�氶亷(gu貌)闆㈠績鐨勬柟寮忛��(j矛n)琛�锛屼竴鏄偤浜嗘洿濂界殑鎺у埗娑查珨寰�Tip涓殑娴佸嚭閫熷害锛堜互涓婅堪5 mgSPE-Ti-IMAC Tip鏌辩偤渚嬶紝涓婃ǎ鍜屾礂鑴殑閬�(gu貌)绋嬮洟蹇冨姏涓�鑸帶鍒跺湪80-100 g锛屾礂婊岄亷(gu貌)绋嬫帶鍒跺湪200 g锛夛紝浜屾槸鍙互淇濊瓑姊濅欢鐨勪竴鑷�锛屽緸鑰屼繚璀夌祼(ji茅)鏋滅殑閲嶇従(xi脿n)鎬с�備笂杩�2-5姝ラ涓殑鏅�(sh铆)闁撶偤闆㈠績鏅�(sh铆)闁�銆�