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培養(yǎng)方法

傳代方法 將舊培養(yǎng)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(yǎng)皿，細胞剛有脫落時，則吸除大部分胰酶，留約0.5mL，移至培養(yǎng)箱消化，約2min取出。傳代用12mL CM1-1培養(yǎng)液終止消化，輕輕吹打均勻細胞，后可分3~6皿培養(yǎng)；

生長條件 37℃，5%CO2，CM1-1培養(yǎng)液。CM1-1培養(yǎng)液：90%DMEM-H+10%FBS。DMEM-H：DMEM高糖培養(yǎng)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。

培養(yǎng)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???入6孔培養(yǎng)板（每孔一片）或培養(yǎng)皿中（每個平皿可放置2-3片）； 

3．在距離紫外燈直射范圍內(nèi)20-30 厘米處照射2-3小時； 

4．將經(jīng)過計數(shù)的細胞懸浮液移入培養(yǎng)板中，使蓋玻片完全浸在培養(yǎng)液中； 

5．將培養(yǎng)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(yǎng)板底部2/3面積時，將培養(yǎng)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。 

實驗要點及說明：

1．本方法適用于貼壁細胞培養(yǎng)，而不適用于懸浮細胞培養(yǎng)，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(jīng)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(tài)一致的細胞，可以使用較大的培養(yǎng)皿，但不宜過大，以避免培養(yǎng)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。

細胞培養(yǎng)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(yǎng)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(yǎng)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(yǎng)基終止消化；若細胞還是貼壁，放回培養(yǎng)箱繼續(xù)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(yǎng)基重懸后加入培養(yǎng)瓶或皿中，T25培養(yǎng)瓶加6-8ml培養(yǎng)基；

⑥懸浮細胞直接離心收集，細胞沉淀重懸后分到新培養(yǎng)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(yǎng)基混勻。

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

3、細胞凍存：待細胞生長狀態(tài)良好時進行細胞凍存保種

①棄去培養(yǎng)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(yǎng)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。

 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(chǎn)品介紹 The cGMP specific phosphodiesterase type 5A (PDE5A) family is comprised of 2 genes (PDE5A and PDE5B) each with multiple splice variants generated by RNA splicing and use of alternate initiation sites. The PDE5 is highly expressed in aorta and lungs, intestine, kidney adrenal gland cerebellum and cerebrum. In cerebellum the PDE5 is highly expressed during neonatal development in Purkinge cells layer. The PDE5 is also abundant in vascular smooth muscle regulating cGMP levels and vascular smooth muscle tonicity. In corpus cavernosum inhibition of PDE5 by sildenafil corrects erectile dysfunctions. The nitric oxide donor sodium notropurside (SNP) stimulate PDE5 activity by cGMP dependent kinase phosphorylation. PDE5A1 has 875 amino acids (99.5 kDa). The amino terminal 142 amino acid of the PDE5 gene showed no sequence homology with other PDEs and also contained serine 92 that is phosphorylated by cGMP kinases.

Function : Plays a role in signal transduction by regulating the intracellular concentration of cyclic nucleotides. This phosphodiesterase catalyzes the specific hydrolysis of cGMP to 5'-GMP.

Tissue Specificity : Expressed in aortic smooth muscle cells, heart, placenta, skeletal muscle and pancreas and, to a much lesser extent, in brain, liver and lung.

Post-translational modifications : Phosphorylation is regulated by binding of cGMP to the two allosteric sites (By similarity). Phosphorylation by PRKG1 leads to its activation.

Similarity : Belongs to the cyclic nucleotide phosphodiesterase family.

Contains 2 GAF domains.

Database links : UniProtKB/Swiss-Prot: O76074.2

英文名稱  Anti-PER1 protein

中文名稱  節(jié)律抑制蛋白PER1抗體

別    名  Circadian clock protein PERIOD 1; Circadian clock protein PERIOD1; Circadian pacemaker protein Rigui; hPER 1; hPER; hPER1; KIAA0482; MGC88021; PER 1; PER; PER1; PER1 protein; PER1_HUMAN; Period 1; Period circadian protein homolog 1; Period drosophila homolog of; Period homolog 1; Period1; RIGUI.

濃    度  1mg/1ml

規(guī) 格  0.2ml/200μg

抗體來源  Rabbit  

克隆類型  polyclonal
非必需氨基酸庚型肝炎病IgG抗體檢測試劑盒對氟苯甲酸 98%醋酸氯己定 98%

C1q腫瘤壞死因子相關蛋白12檢測試劑盒對氟苯甲酸 99%,升華純化鹽酸洗必泰 96%

表皮生長因子受體抗體檢測試劑盒3-氟苯甲酸 98%薯蕷皂素 分析標準品，>95%

周期素依賴性激酶4檢測試劑盒2-乙酰噻吩 99%薯蕷皂素 90%

凝血因子ⅩI檢測試劑盒3,4-二甲氧基 98%薯蕷皂素 95%

表皮生長因子受體2抗體檢測試劑盒噻吩-2- 97%氘代酮 (D,99.8%) + TMS (0.03%)

血清特異性IgE抗體檢測試劑盒2-噻吩乙 98%氘代酮 (D,99.9%)