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上海乾思生物科技有限公司
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免疫沉淀|實驗技術服務

時間：2015/8/1閱讀：314
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關鍵詞:免疫沉淀|實驗技術服務
簡介：世界*品牌免疫沉淀|實驗技術服務原裝,，質量保證,*,。
免疫沉淀|實驗技術服務——pCzn1質粒+Arctic Express宿主菌低溫表達體系。
我們具備豐富的蛋白表達設計經驗及實驗操作技巧,，承諾客戶不成功不收費,。我們所有的實驗數(shù)據(jù)真實可信，我們提供原始的表達菌株和克隆質粒,，客戶可以依據(jù)我們的實驗報告重復我們的實驗結果,。
產品品牌：   http://www.。,。ｃｏｍ/goodsid/fenleiyi/2868603/1.html    
測序實驗流程：
材料
1,，  蛋白A 或蛋白G
2,，  一抗
3，  免疫沉淀緩沖液：20 mM 磷酸鈉, pH 7.5, 500 mM NaCl, 0.1% SDS, 1% NP40, 0.5% 脫氧膽酸鈉 and 0.02% *.
（> NOTE: 50 mM 醋酸鈉緩沖液, pH 5.0,. 500 mM NaCl, 0.1% SDS, 1% NP-40 and 0.02%* 也可作為免疫沉淀的緩沖液,，能提高蛋白G的結合功效）
4,，  洗脫液：0.1 M 甘氨酸-HCl buffer, pH 2.5
5，  SDS-PAGE 上樣緩沖液 (pH 6.8)： 2% SDS, 62.5 mM Tris 堿, 10% 甘油, 2-5% ,
步驟：
1,，  用50 μl免疫沉淀緩沖液溶解抗原,，向溶液中加入1倍過量的特異性一抗。加入免疫沉淀緩沖液至樣品體積為0.2 ml,。一般情況下,，在此體積下2-5μg的純化抗體能夠較好地形成抗原抗體復合物。
2,，  樣品4°C.孵育,。
3，  將適量的蛋白A或蛋白G加到抗原抗體復合物中(~ 50 μl of gel per 5 μg of antibody),。
4,，  室溫下，輕柔混勻樣品,，孵育2小時,。
用0.5ml免疫沉淀緩沖液洗蛋白A或蛋白G連接的抗原抗體復合物，離心2-3分鐘,，棄上清,。重復此步驟至少6次。
5,，  用50 μl洗脫液孵育蛋白A或蛋白G連接的抗原抗體復合物5分鐘,，將抗原抗體復合物從蛋白A或蛋白G上洗脫下來，離心,，收集上清,。另用50 μl洗脫液孵育膠5分鐘，離心,，收上清,。將兩個上清混合。
6,，  立即調節(jié)蛋白樣品的pH至生理值,，用一種合適的、多次離心的緩沖液調節(jié),，如1.0 M Tris, pH 7.5 (10 μl of this buffer to 100 μl of the supernatant should be sufficient).
7,，  將洗脫成分除鹽。
8，  準備好樣品待定性,。
NOTE：如果用SDS-PAGE定性蛋白質,，省去步驟6-9。在完成第5步之前,，用0.5ml蒸餾水洗膠,。離心蛋白A或蛋白G 2-3分鐘，棄上清,。用25μl SDS-PAGE上樣緩沖液在95°C孵育蛋白A或蛋白G連接的抗原抗體復合物5分鐘,。樣品離心，收集上清,。重復一次,，匯集上清至總體積50μl。由此樣品則準備妥當,。

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