基于细胞的酶联免疫检测实验
ImmunoWay
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基于细胞的酶联免疫检测实验(Cell Based Elisa)可以:
(1)检测高通量的样本,定量细胞膜上或细胞内的蛋白质的表达情况。
(2)用于磷酸化蛋白质的检测,易降解蛋白质的检测,少量细胞目的蛋白质的检测。
试验方法原理
先将细胞种于 96 孔板中,对细胞进行不同条件的处理。待检测目的蛋白时,对细胞进行多聚甲醛的固定、灭活、打孔及封闭非特异性结合位点。根据抗原抗体反应的原理,一抗孵育与目的蛋白质结合,加入酶标记二抗与一抗结合,加入底物显色后酶标仪读取数值。
实验材料
细胞
细胞
试剂、试剂盒: 多聚甲醛 Cell Based Elisa 试剂盒
仪器耗材: 移液枪 酶标仪 恒温箱
实验步骤
1 将细胞种于 96 孔培养板中。
2 培养细胞,根据实验设计对细胞进行不同条件的处理。
3 待检测目的蛋白时,对细胞进行多聚甲醛的固定、灭活、打孔及封闭非特异性结合位点。加入一抗孵育与目的蛋白质结合,加入酶标记二抗与一抗结合,加入底物显色后酶标仪读取数值。
4 细胞清洗后加入结晶紫与细胞核结合读取数值,作为内参值进行比对。
5 数据处理。
注意事项
1 根据细胞的大小和生长情况进行种板和培养,待检测时细胞的密度控制在 75-90%。
2 对于悬浮细胞,加入 100ul 10ug/ml 的多聚-L-赖氨酸,37℃ 的条件下处理 30 min 将悬浮细胞贴壁到培养板上,PBS 清洗两次,每次 5 min。
3 若是悬浮细胞,加入 100ul 的 8% 多聚甲醛室温孵育 25-30 min 将细胞固定。
4 甲醛具有挥发性,终止液具有腐蚀性,请带好防护设备。
优势
1 样本的制备过程中,不涉及裂解细胞,只需将细胞及蛋白质原位固定,降低蛋白质发生降解的可能性。
2 在操作过程中,不涉及传统免疫印迹法的制胶,跑胶,转膜,曝光等步骤,为研究人员节省很多时间。
3 整个操作在 96 孔板中进行,容易实现高通量样本的检测,或者少量细胞目的蛋白质检测。
4 操作简单,很容易实现多个平行,多次重复,所得数据具有统计分析意义。
应用 Cell Based Elisa 发表的文章:
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5 Sanjiv Dhingra, Ashim K. Bagchi, Ana L. Ludke, Anita K. Sharma, Pawan K. Singal. Akt Regulates IL-10 Mediated Suppression of TNFa- Induced Cardiomyocyte Apoptosis by Upregulating Stat3 Phosphorylation. PLoS ONE 2011; 6(9): e25009.
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9 Abderrahim El Guerrab1, Rabah Zegrour, Carine-Christiane Nemlin, Flavie Vigier, Anne Cayre, Frederique Penault-Llorca3, et al. Differential Impact of EGFR-Targeted Therapies on Hypoxia Responses: Implications for Treatment Sensitivity in Triple-Negative Metastatic Breast Cancer. PLoS ONE 2011; 6(9): e25080.
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