Biotinylated Recombinant SARS-CoV-2 Spike S1-Fc-Avi Protein
Source: Biotinylated recombinant SARS-CoV-2 Spike S1 protein (Gln14-Arg683) was expressed in mammalian cells with a His tag and Avi at the C-terminus.Accession: QHD43416.1Predicted molecular mass: 77.9 kDa. Due to glycosylation, the biotinylated recombinant SARS-CoV-2 Spike S1 protein migrates to 110-120 kDa based on the Bis-Tris PAGE result.Endotoxin: Less than 1 EU per ug by the LAL method.Activity: Immobilized biotinylated recombinant SARS-CoV-2 Spike S1-His-Avi protein at 1 ug/ml (100 ul/well). Dose response curve for the recombinant human ACE2-Fc fusion protein with the EC50 of 81 ng/ml determined by ELISA.Formulation: The biotinylated recombinant SARS-CoV-2 Spike S1-His-Avi protein was lyophilized from 0.22 um filtered solution in 20 mM PB (pH 7.4). Normally 5% trehalose is added as protectant before lyophilization.Purity: > 95% by PAGE under reduced condition, and SEC-HPLC.Shipping: The product is shipped with ice packs. Upon receipt, store it immediately at the temperature recommended below.Stability & Storage:Use a manual defrost freezer and avoid repeated freeze-thaw cycles.12 months from date of receipt, -20 to -70°C as supplied.1 month, 2 to 8°C under sterile conditions after reconstitution.3 months, -20 to -70°C under sterile conditions after reconstitution.
Background
The spike protein (S) of coronavirus (CoV) attaches the virus to its cellular receptor, angiotensin-converting enzyme 2 (ACE2). A defined receptor-binding domain (RBD) on S mediates this interaction. The S protein plays key parts in the induction of neutralizing-antibody and T-cellresponses, as well as protective immunity.
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产生这一现象的原因在于 DNA合成酶只能沿5'-3'的方向合成DNA 而DNA本身的两条链又是反向分布的 所以就造成了只有一条链合成可以连续地进行下去（以它为模板的子链生成方向正好是DNA聚合酶可以直接提供的） 而后随链要盘绕成回环 反扭过来 才能合成
再合成起始的时候 DNA聚合酶是需要一段RNA引物的 在原核生物中这一引物是由dnaQ(一种酶)在已解旋的单链5'端合成，真核生物中也有对应的酶 由于后随链的合成不连续 所以每个片段都要有引物 在DNA合成结束的时候 这些引物要被切除 因而留下缺口 这时又要特定的酶去填补缺口（比如 大肠杆菌中的DNA聚合酶I）可是填补序列和周围序列间会有缺刻 也就是说他们交界处的3'-5'磷酸二脂键是断开的 这时需要DNA连接酶发挥作用 将其连好
所以 后随链上的缺刻多 还真够DNA连接酶忙一阵的 前导链上只有一开始有RNA引物 因此 最后也基本只有这个地方会用到连接酶
DNA连接酶主要是连接DNA片段之间的磷酸二酯键最初从原核生物（大肠杆菌）分离得到的.现在生物基因工程主要是从T4噬菌体中分离得到的,
大家有用过Invitrogen的T4连接酶吗？说明书上是23－26度连接，一般的连接酶不都是16度吗？应该用多少度呢？另外，说明书上还说连接后为了达到更好的转化效率，应将连接反应液至少稀释5倍再转化，是这样吗？谢谢大家帮忙啊
求有经验的大神指教，我的载体和目的基因连不上，转化不到大肠中，比例为1:3。另外，为啥胶回收后的载体浓度那么低，大约6ng/ul了，影响连接么？
有没有人用过NEB的Blunt/TALigaseMasterMix和InstantSticky-endLigaseMasterMix？
有没有人用过NEB的Blunt/TALigaseMasterMix和InstantSticky-endLigaseMasterMix？
有没有人用过NEB的Blunt/TALigaseMasterMix和InstantSticky-endLigaseMasterMix？
.....它说连得又快又好，想问问有木有小白鼠试过它家的这两个产品呢。。如果用过能不能请教下效果咋样呢。。我之前用Thermo的T4，属于又便宜又慢效果还可以的那类型的。。（虽然说thermo也说室温30min就ok，但是还是4度过夜了，室温30min不靠谱）
只不过限制性核苷酸酶是将磷酸二酯键切断；而DNA连接酶则是形成磷酸二酯键。
