怎么分離抗體和與抗體連接好的材料
將抗體與我們制備的材料(~50nm)用的EDC和NHS連接,最后怎樣才能把沒連上抗體的洗掉,得到比較純的連接物呢? the free non-conjugated QDs and byproduct isourea were removed by ultrafiltration using 100 K Nanosep centrifugal devices by centrifugation at 5000 rmp for 15 min.The lower phase, contain-ing free QDs and isourea, were discarded. To wash the conju-gate and reduce the impurities, the upper phase, containing QD-IgG, was diluted by 300 L PBS buffer, and additionally subjected to centrifugation at 5000 rmp for 15 min. This washing step was repeated twice. The conjugate was recovered by 100 L of PBS and transferred to a new Eppendorf tube and then stored at 4 °C in a dark until use. 實驗剛起步,覺得ODs-lgG的大小跟ODs的差不多耶,因該混在一起吧,不理解文獻中的方法。謝謝各位師兄師姐指點~
返回小木蟲查看更多
京公網安備 11010802022153號
什么試劑呀?
這個要看量子點的粒徑大小和在溶液體系中的分散性如何了。
可以試試通過簡單離心的方法,蛋白在溶液,4000~5000rpm的離心速度不容易離心下去,如果量子點能夠離下去,就能分離開了,然后對離心物進行清洗幾次,把結合不牢的洗去就OK了。
謝謝啦^_^
QDs下去了,那么ODs-抗體的也會下去吧~這樣怎么分離它們倆呢
,
額,如果比例合適的話,QDs表面基本上都標記了吧,不知道你還做不做封閉?
我們不是用的QDs,是用的上轉換連的是氨基,原理差不多嘛。不懂文獻那種方法是怎么分離滴。
他們進行了超濾分離, "the free non-conjugated QDs and byproduct isourea were removed by ultrafiltration using 100 K Nanosep centrifugal devices by centrifugation at 5000 rmp for 15 min" 也就是說用離心的方法,離心管是套式的(大離心管里面有小離心管,中間有濾膜的那種),這個跟做PCR之前的核酸提取方法差不多,標記好的QDs留在了上層里,free的濾到了下層(The lower phase, contain-ing free QDs and isourea, were discarded.)。