在做Western Blot的时候,膜的选择是整个实验的一个关键点,所以大家要谨慎选择。毕竟它是一个物理支撑物,不但要hold住宝贵的样品,还要有“坚韧不拔”的精神来抵抗住各种实验条件。每个研究人员的目标蛋白特点和下游检测方法不尽相同,要想找到适合自己的转渍膜,需要通过一系列的实验来找到。为了方便大家比较和参考,小编在这里对比和总结了Nitrocellulose 和PVDF膜的特点(如下表所示)。如果大家有更多建议,也欢迎和我们取得联系。
表格1:Nitrocellulose 和 PVDF 性质比较。
|
Nitrocellulose |
PVDF |
中文名称 |
硝酸纤维素膜 |
聚偏二氟乙烯膜 |
与蛋白质的结合能力(μg/cm2) |
80-100 |
170-200 |
详细用途 |
·Western blotting (尤其针对分子量小于20kDa的蛋白质) ·核酸分析 ·氨基酸分析 ·Dot/slot blotting |
·Western blotting (尤其针对分子量较大的蛋白质) ·蛋白质测序 ·氨基酸分析 |
与蛋白质结合的作用力 |
疏水性相互作用 |
疏水性相互作用和偶极–偶极相互作用 |
物理特性 |
相对脆弱 |
较高的机械强度 |
膜孔径的基本信息 |
是的。产品通常都有0.1, |
|
化学耐性 |
没有 |
有 |
敏感度 |
敏感程度相对较低,但是可以大大减少背景信号。 |
可以检测蛋白质含量非常低的样品,但同时也会有很多背景信号作为干扰。 |
蛋白质剥离并重新探测的过程 |
不建议。信号会大大减弱。 |
可以。蛋白质在膜上的吸附程度更紧密。 |
甲醇的使用 |
需要。转渍过程中必须使用。但是可以导致大分子量的蛋白质沉淀。 |
不需要。膜必须在使用之前用甲醇浸泡,但在转渍过程中可以使用不含有甲醇的缓冲液。这个方法可以预防分子量较大的蛋白质沉淀。 |
SDS的添加 |
不需要。这样可以增大小分子量的蛋白质与膜结合。 |
需要。这样可以大大提高转渍效率。 |
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