Antibiotics for the selection and maintenance of eukaryotic cells that are stably transfected with vectors expressing neomycin, puromycin, and hygromycin resistance cassettes.
Antibiotics for the selection and maintenance of eukaryotic cells that are stably transfected with vectors expressing neomycin, puromycin, and hygromycin resistance cassettes.
G418, Puromycin, & Hygromycin B dose concentrations for selection & maintenance
Antibiotic | |||
G418 | |||
Puromycin | |||
Hygromycin B |
For selecting colonies, the appropriate dose must be determined empirically for each specific cell line. We recommend testing a dosage range using dishes of untransfected cells and choosing the dose that kills all of the cells in 3–5 days. If all of the cells die in less than 24 hours, you should use a lower dose.
ebiomall.com
>
>
>
>
>
>
>
>
>
>
>
>
血清内含有抗体,封闭的时候,选用同物种的血清,比如,使用小鼠单抗的话,就选用小鼠血清来进行封闭。这样,如果细胞表面有可以非特异性结合小鼠抗体的部位都会事先被结合,就不会再结合荧光标记的特异性抗体了。
如果一个抗体可以用来“做western blot,免疫荧光,免疫共沉淀,ELISA”,我估计着是HRP或碱性磷酸酶或生物素之类标记的,然后需要再加荧光标记的二抗,这类抗体也是不能做流式用的。
我只用过流式的抗体来做免疫荧光(直接染色),效果还不错,但是反过来就没试过。
比如,你使用了小鼠IgG1, PE 标记的特异性抗体,那就选用小鼠IgG1, PE标记的非特异性抗体。
下面这个图,是一个比较典型的使用了同型对照的结果图向左转|向右转灰色的曲线是未染色的样本,黄色是同型对照,红色是特异性抗体染色。由于细胞对抗体的非特异性结合,一般同型对照都会比未染色样本的荧光信号要强。所以设置阴性阈值的时候,要根据同型对照,而不是未染色的细胞。