實驗要點及說明：

1．本方法適用于貼壁細胞培養，而不適用于懸浮細胞培養，懸浮細胞可使用滴片法； 

2．所使用的蓋玻片應該為玻璃**，并經過鉻酸洗液處理； 

3．蓋玻片非常薄，易碎，取放蓋玻片時動作要輕； 

4．如果需要更多生長狀態一致的細胞，可以使用較大的培養皿，但不宜過大，以避免培養液的浪費和增加污染機率； 

5．如果細胞貼壁生長能力較差，可將蓋玻片在0.5%多聚賴氨酸溶液中浸泡5-10分鐘并自然晾干。

培養方法

傳代方法 將舊培養液吸除，PBS清洗兩遍后，加入6mL（/100mm皿）胰酶，在顯微鏡下觀察，期間禁止搖晃培養皿，細胞剛有脫落時，則吸除大部分胰酶，留約0.5mL，移至培養箱消化，約2min取出。傳代用12mL CM1-1培養液終止消化，輕輕吹打均勻細胞，后可分3~6皿培養；

生長條件 37℃，5%CO2，CM1-1培養液。CM1-1培養液：90%DMEM-H+10%FBS。DMEM-H：DMEM高糖培養液，含谷氨酰胺，含丙酮酸鈉。

存儲條件 凍存則用6mL凍存液（90%FBS+10%DMSO）終止消化，吹打均勻，分為6支凍存管，用程序降溫盒于-80℃凍存，過夜轉移至液氮中保存。

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培養操作步驟 ：

1．用蓋片鑷將蓋玻片自75%乙醇中取出，用無菌絲綢布擦拭干凈，不要用紗布； 

2．將蓋玻片輕輕放入6孔培養板（每孔一片）或培養皿中（每個平皿可放置2-3片）； 

3．在距離紫外燈直射范圍內20-30 厘米處照射2-3小時； 

4．將經過計數的細胞懸浮液移入培養板中，使蓋玻片完全浸在培養液中； 

5．將培養板在5% CO2水浴孵箱中37℃孵育2-3天，當貼壁細胞生長至覆蓋培養板底部2/3面積時，將培養板取出，用蓋片鑷輕輕取出蓋玻片，用蒸餾水漂洗后即可進行快速固定以及細胞化學檢測。 

細胞培養方法：

1、細胞傳代：細胞密度達到80-90%時即可傳代

①棄去培養上清，用PBS或生理鹽水清洗1-2次；

②加入2ml0.25%胰酶（T25瓶），使胰酶覆蓋整個瓶或皿，蓋好放入培養箱消化；

③1-2min后，顯微鏡下觀察細胞，若大部分細胞回縮且有少量細胞脫落，輕輕吹打下確認消化情況后加入完全培養基終止消化；若細胞還是貼壁，放回培養箱繼續消化至可以輕輕吹打下為止；

④將細胞懸液1000RPM左右條件下離心4min，棄上清；

⑤用新鮮培養基重懸后加入培養瓶或皿中，T25培養瓶加6-8ml培養基；

⑥懸浮細胞直接離心收集，細胞沉淀重懸后分到新培養瓶中。

2、細胞復蘇：

①將凍存管在37℃溫水中快速搖晃融化，時間1min左右，加入4-5ml培養基混勻。

②在1000RPM左右條件下離心4min，棄上清,加1-2ml培養基吹勻，將細胞懸液加入培養瓶中，補加適量培養基。

3、細胞凍存：待細胞生長狀態良好時進行細胞凍存保種

①棄去培養上清，用PBS或生理鹽水清洗1-2次，加入1mL 0.25%胰蛋白酶（T25瓶）

②1-2min后，顯微鏡下觀察細胞，大部分細胞回縮且有少量細胞脫落，輕輕吹打下確認消化情況后加入完全培養基終止消化；

③將細胞懸液1000RPM左右條件下離心4min，棄上清，加1ml凍存液重懸細胞；

④將凍存管放入程序降溫盒，放入-80℃冰箱，4小時后將凍存管轉入液氮罐儲存。

研究領域  學 染色質和核信號 信號轉導 激酶和磷酸酶  

蛋白分子量  predicted molecular weight: 42kDa

性    狀  Lyophilized or Liquid

免 疫 原  KLH conjugated synthetic peptide derived from human PP2CA/PPM1A

亞    型  IgG

純化方法  affinity purified by Protein A

儲 存 液  0.01M PBS, pH 7.4 with 10 mg/ml BSA and 0.1% Sodium azide

產品應用   WB=1:100-500  ELISA=1:500-1000  IP=1:20-100  IHC-P=1:100-500  IHC-F=1:100-500  IF=1:100-500

（石蠟切片需做抗原修復）

 not yet tested in other applications.

 optimal dilutions/concentrations should be determined by the end user.  

保存條件  Store at -20 °C for one year. Avoid repeated freeze/thaw cycles. The lyophilized antibody is stable at room temperature for at least one month and for greater than a year when kept at -20°C. When reconstituted in sterile pH 7.4 0.01M PBS or diluent of antibody the antibody is stable for at least two weeks at 2-4 °C.

Important Note  This product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications.

產品介紹 The protein encoded by this gene is a member of the PP2C family of Ser/Thr protein phosphatases. PP2C family members are known to be negative regulators of cell stress response pathways. This phosphatase dephosphorylates, and negatively regulates the activities of, MAP kinases and MAP kinase kinases. It has been shown to inhibit the activation of p38 and JNK kinase cascades induced by environmental stresses. This phosphatase can also dephosphorylate cyclin-dependent kinases, and thus may be involved in cell cycle control. Overexpression of this phosphatase is reported to activate the expression of the tumor suppressor gene TP53/p53, which leads to G2/M cell cycle arrest and apoptosis. Three alternatively spliced transcript variants encoding distinct isoforms have been described. [provided by RefSeq, Jul 2008].

Function : Enzyme with a broad specificity. Negatively regulates TGF-beta signaling through dephosphorylating SMAD2 and SMAD3, resulting in their dissociation from SMAD4, nuclear export of the SMADs and termination of the TGF-beta-mediated signaling.

Subunit : Monomer. Interacts with SMAD2; the interaction dephosphorylates SMAD2 in its C-terminal SXS motif resulting in disruption of the SMAD2/SMAD4 complex, SMAD2 nuclear export and termination of the TGF-beta-mediated signaling. Interacts with SMAD2; the interaction dephosphorylates SMAD2 in its C-terminal SXS motif resulting in disruption of the SMAD2/SMAD4 complex, SMAD2 nuclear export and termination of the TGF-beta-mediated signaling.

Subcellular Location : Nucleus.

Similarity : Belongs to the PP2C family.
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