One Step PrimeScript III RT-qPCR Mix is a 2X master mix for one-step real-time, probe-based RT-qPCR (using the 5′ nuclease method). It contains a novel heat- and inhibitor-resistant reverse transcriptase, PrimeScript III RTase, in combination with an efficient, specific hot-start PCR enzyme, TaKaRa Taq HS, and an optimized reaction buffer. A quick and simple protocol allows the reverse transcription and qPCR reactions to be performed in the same tube, reducing the risk of contamination.
One Step PrimeScript III RT-qPCR Mix is a 2X master mix for one-step real-time, probe-based RT-qPCR (using the 5′ nuclease method). It contains a novel heat- and inhibitor-resistant reverse transcriptase, PrimeScript III RTase, in combination with an efficient, specific hot-start PCR enzyme, TaKaRa Taq HS, and an optimized reaction buffer. A quick and simple protocol allows the reverse transcription and qPCR reactions to be performed in the same tube, reducing the risk of contamination.
PrimeScript III RTase displays increased heat tolerance (up to 55°C) while maintaining the specificity and robustness of PrimeScript RT. This allows cDNA synthesis from RNA with a more complex secondary structure. It is also highly resistant to a wide variety of inhibitory substances such as heparin (blood) and humic acid (soil), allowing stable one-step real-time RT-qPCR to be performed on a wide variety of samples. While One Step PrimeScript III RT-qPCR Mix often works well with crude RNA extracts, using a highly pure RNA sample is recommended if the target sequence is not detectable or if more accurate, stable analysis results will be needed. It can be used for various applications such as gene expression and RNA virus detection, and it is well-suited for multiplex real-time PCR.
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1、如果是提取的总蛋白，然后做WB，用β-actin或者GAPDH做内参肯定是没有问题的，这是公认的东西。
2、如果用膜蛋白提取试剂盒提取蛋白，再用β-actin作为内参似乎不妥，因为理论上来讲β-actin在膜上是不表达的。WB能做出β-actin来是因为膜蛋白提取时把胞质蛋白也提出来了。然而，如果我们试验目的是用药物处理细胞，比较处理前后某种膜蛋白的表达情况，此时用膜蛋白提取试剂盒提取膜蛋白后再用β-actin做内参似乎就不妥了，因为你根本不知道药物处理前后混杂了多少的胞质蛋白进来。如果没有胞质蛋白混进去的话，β-actin就是检测不到的。
1. 使用预染 Marker，不过分子量不是特别准；
2. 所使用的 Marker 条带与待测蛋白质带有相同的抗原表位，比如都带有 His 融合标签；
3. 可以使用普通的 Marker 转膜使用 丽春红 等染色，然后在膜上标记出 Marker 各条带的位置。
原液分装。抗体孵育之前稀释。
做WB的浓度不一定需要很高的。可以设置简单梯度1:200；1:500；1:1000 。看结果再优化
