Obtain a rapid burst of Cre expression in mammalian cells with footprint-free delivery of Cre mRNA in nonintegrating, virus-like particles. Cre RNA lentiviral particles (RLPs) contain no viral genome and are generated using a novel packaging technology that loads multiple biologically active mRNAs per particle. RLPs have been shown to mediate transient protein expression at high efficiency for in vitro and in vivo applications involving cell lines, primary cells, stem cells, tissues, and organisms.
Obtain a rapid burst of Cre expression in mammalian cells with footprint-free delivery of Cre mRNA in nonintegrating, virus-like particles. Cre RNA lentiviral particles (RLPs) contain no viral genome and are generated using a novel packaging technology that loads multiple biologically active mRNAs per particle. RLPs have been shown to mediate transient protein expression at high efficiency for in vitro and in vivo applications involving cell lines, primary cells, stem cells, tissues, and organisms.
Popular methods for Cre recombinase-based genome editing present tradeoffs. Transient transfection of a plasmid or mRNA limits the scope of Cre activity, minimizing the likelihood of unwanted effects associated with persistent Cre expression, but is associated with cellular toxicity, is unsuitable for in vivo applications, and is only feasible for cells that can be readily transfected. Transduction of the Cre gene sequence on a viral vector enables Cre expression in a broader range of cell types and can be applied to in vivo studies, but is associated with random genomic integration, may require activation and/or selection of target cells, and may result in undesirable persistence of Cre expression.
Cre recombinase RLPs enable researchers to overcome the above limitations and express Cre more rapidly and efficiently in mammalian cells by delivering nonviral Cre mRNA in lentivirus-derived packaging in the absence of a viral genome. In vitro studies involving RLP-mediated delivery of a luciferase reporter demonstrated that reporter expression was maximal at 4–24 hours post application and had declined rapidly at 30 hours. By contrast, expression of luciferase delivered via transfection was undetectable until 8 hours and peaked at 24 hours, while expression via lentiviral integration was undetectable until 24 hours. In an in vivo study involving a loxP mouse line (ROSA26-YFP), local injection of Cre RLPs into muscle yielded a zone of reporter gene expression that was comparable in size to a zone generated via injection of purified Cre lentivirus (Prel et al. 2015).
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没有marker，怎么知道你做的蛋白大小？
没有参照物，怎么知道你跑的快不快？
没有尺子，怎么知道你的size大小？
凭嘴说吗？
抗体的特异性鉴定 抗体的特异性是指与相应抗原或近似抗原物质的识别能力。抗体的特异性高，它的识别能力就强。衡量特异性通常以交叉反应率来表示。交叉反应率可用竞争抑制试验测定。以不同浓度抗原和近似抗原分别做竞争抑制曲线，计算各自的结合率，求出各自在 IC50时的浓度，并按下列公式计算交叉反应率。 如果所用抗原浓度IC50浓度为pg/管，而一些近似抗原物质的IC50浓度几乎是无穷大时，表示 这一抗血清与其他抗原物质的交叉反应率近似为 0，即该血清的特异性较好。
抗体的亲和力 是指抗体和抗原结合的牢固程度。亲和力的高低是由抗原分子的大小、抗体分子的结合位点与抗原决定簇之间立体构型的合适度决定的。有助于维持抗原抗体复合物稳定的分子间力有氢键、疏水键、侧链相反电荷基因的库仑力、范德华力和空间斥力。亲和力常以亲和常数K表示，K的单位是L/mol，通常K的范围在 108 ～1010 /mol，也有多达 1014 /mol。抗体亲和力的测定对抗体的筛选，确定抗体的用途，验证抗体的均一性等均有重要意义。向左转|向右转
不过多克隆抗体因为有多个结合位点，事实上的特异性并不如单克隆抗体，所以一般Western blot都尽量要求使用单克隆抗体。以保证实验结果准确可靠
一般来说多克隆的阳性率高一些，但出现假阳性的比例也高一些。
又由于自然存在的抗原大都存在多个抗原表位,会刺激机体产生多种针对同一抗原的不同抗原表位相应的不同抗体.
1取组织
（1） 灌注固定：暴露心脏,将注射针头插入左心室,剪开右心耳,快速灌注PBS,待右心耳处流出澄清液体,换4%多聚甲醛灌注到大鼠四肢强直为止,快速取脑和肾。
（2） 固定：将组织置于4%多聚甲醛中固定15小时
（3） 脱水：将组织放入30% 蔗糖溶液中直至下沉
（4） 切片：脑组织冰冻切片20微米，37度烘干2h以上；
（5）将切片放置－80度保存，随用随取。
2免疫单标记
（1）做抗原修复（0.4g柠檬酸、3g柠檬酸钠、1000ml水配制成溶液，放到水浴箱中90~94℃保存15min）,冷却后， 用1*PBS反复洗标本10min，吸去PBS，同时向标本加渗透液（配0.4%trition液，然后将BSA加入到配好的trition液中，配成1％浓度）100ul，常温下湿盒反应1h。
（2） 吸去渗透液，同时向标本加block液（5％封闭用正常山羊血清）100ul，常温下湿盒反应5h。
（3） 吸去block液，同时向标本加入1：50稀释的羊抗鼠一抗100ul，37度湿盒反应过夜。
（4）吸去一抗，并回收，用1*PBS洗液反复洗15min。
（5）快速从冰箱中取出驴抗羊的二抗，并立即用锡纸包裹，注入到一个新的以锡纸包裹的tube管中并用PBS以1：200稀释。
（6）进入暗室，吸去PBS，同时加入二抗100ul，37度湿盒反应2h,封片。
（7）放置到荧光显微镜下观察。
