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View additional product information for Mammalian Lumio™ Gateway® Vectors, with Dual Lumio™ Red and Green In-Cell Detection Kit - FAQs (12589032)
11 product FAQs found
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.