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查看更多产品信息 Mammalian Lumio™ Gateway® Vectors, with Dual Lumio™ Red and Green In-Cell Detection Kit - FAQs (12589032)
20 个常见问题解答
在BP/LR Clonase反应的一步法实验方案中，不建议用BP Clonase酶和LR Clonase酶替代BP Clonase II 酶/LR Clonase II酶，因为这样的重组效率非常低。
有的，我们能提供针对BP/LR Clonase反应的一步式实验方案DNA可以在一步反应后被克隆到目的载体中，从而节省了您的时间和金钱。
建议使用一个供体载体进行一次BP反应以获得一个入门克隆。然后将这一入门克隆和目的载体进行一次LR反应以获得新的表达克隆。
5X LR Clonase缓冲液或5X BP Clonase缓冲液不作为单独产品出售。它们作为酶试剂盒的一部分进行销售。
我们不提供任何用于在植物内表达的Gateway载体。
Lumio试剂具有疏水性，因此很容易穿过细胞膜。无需通过透化处理来帮助该试剂进入细胞内部。
哺乳动物细胞培养基中的血清蛋白（如BSA，66KD）可能会与Lumio试剂发生交叉反应，从而形成非特异性的条带。吸去细胞培养基并在收获细胞后使用PBS润洗哺乳动物细胞3-4遍，有助于减少BSA的非特异性结合。
我们尚未在细胞中发现用于蛋白检测浓度的Lumio试剂具有任何不良效应。我们也未在使用Lumio Green之后发现细胞形态出现任何不良改变。加入Lumio Red后，我们确实发现细胞发生了某些微小的形态改变，不过这一变化在加入试剂24小时之后恢复。
Lumio染色技术的优势在于可同时兼容体内和体外的蛋白标记操作。在体内标记实验中，向细胞中加入Lumio试剂后即可在荧光显微镜下观察到细胞/蛋白。这一效果与GeneBLAzer检测步骤相似，只是GeneBLAzer是基于扩增报告基因信号的酶促反应。GFP的荧光信号只能在细胞（体内）中检测到，因为需要GFP蛋白的正确折叠。相比GeneBLAzer检测法中的bla蛋白（264个氨基酸，29 kDa）和GFP蛋白（27 kDa）而言，Lumio标签非常之小（6个氨基酸，585Da），因此在很大程度上不会对所融合蛋白的功能造成影响。GFP的劣势在于需要融合一个很大的标签蛋白，而且该检测法并非是基于酶法的报告系统。不同于GeneBLAzer检测法和GFP标签，Lumio-标签蛋白可在Lumio试剂处理细胞裂解液或蛋白之后通过凝胶电泳变得可视化。与Lumio和GFP相比，GeneBLAzer检测法在活体细胞的应用中更为灵敏。GeneBLAzer检测法也不同于Lumio和GFP，这一方法能够实现比率化的读数，因此有助于减少样本间的差异。
In the single-step protocol for the BP/LR Clonase reaction, we would not recommend substituting the BP Clonase II/LR Clonase II enzymes with BP Clonase /LR Clonase enzymes as this would result in very low recombination efficiency.
Yes, we have come up with a single-step protocol for BP/LR Clonase reaction (http://www.thermofisher.com/us/en/home/life-science/cloning/gateway-cloning.html#1), where DNA fragments can be cloned into Destination vectors in a single step reaction, allowing you to save time and money.
We would recommend performing a BP reaction with a Donor vector in order to obtain an entry clone. This entry clone can then be used in an LR reaction with the Destination vector to obtain the new expression clone.
We do not offer the 5X LR Clonase buffer and 5X BP Clonase buffer as standalone products. They are available as part of the enzyme kits.
We do not offer any Gateway vectors for expression in plants.
The Lumio reagent is hydrophobic and can easily pass through the membrane. There is no need to permeabilize the membrane in order to get this reagent into cells.
Serum proteins such as BSA (66 kDa) from the mammalian cell culture medium may cross-react with the Lumio reagent, producing non-specific bands. Removing the cell culture medium and washing the mammalian cells 3-4 times with PBS after harvesting the cells minimizes the non-specific binding from BSA.
We have not experienced negative effects with Lumio reagents at the concentrations used to detect protein in the cells. We also do not see any change in cell morphology when using Lumio Green. After application of the Lumio Red, we do see some minor morphological changes in the cells that are reversed after 24 hours of application of the reagent.
The advantage of Lumio staining is that one can do both in vivo and in vitro protein labeling. For in vivo labeling, load the cells with the Lumio reagent and then visualize the cells/proteins under a fluorescence microscope. This is similar to the GeneBLAzer detection procedure except that GeneBLAzer detection is based on an enzymatic reaction that amplifies the reporter signal. GFP fluorescence can only be detected within the cell (in vivo) because proper protein folding is needed. The Lumio tag is very small (6 amino acids, 585 Da), in contrast to the bla protein in GeneBLAzer detection (264 amino acids, 29 kDa) and the GFP protein (27 kDa), and therefore most likely will not interfere with the function of the protein it is fused to. GFP has the disadvantage of being a large fusion tag and is not an enzymatic-based reporter system. Unlike GeneBLAzer detection and GFP, a Lumio-tagged protein can be visualized on a gel after treating the cell lysate or protein with the Lumio reagent. Compared to Lumio and GFP, GeneBLAzer detection is a more sensitive detection method for use in live cells. Also unlike Lumio and GFP, the GeneBLAzer detection method allows for ratiometric read-outs and thus eliminates sample-to-sample variation.
Yes, fluorescent protein-expressing cells can be fixed using 4% paraformaldehyde in PBS for 10 min followed by one quick PBS rinse and 3 x 5 min washes with 1 mL PBS.
Yes, all of the fluorescent proteins offered by (EmGFP, YFP, CFP, BFP and Cycle 3 GFP) have been humanized for optimal mammalian expression.