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The luciferase from firefly is the most classic luciferase. This 61KD monomer enzyme is directly involved in enzyme activity without modification. The catalytic reaction is dependent on ATP. It is also very suitable as a genetic reporter gene. It is also known as Renilla. Luciferase, only 36KD protein, the substrate is coelenterazine, only requires oxygen, does not require ATP, emits blue light, has been a substitute for firefly luciferase, and often combined with firefly luciferase to become a two-color detection Or as an internal control.
1. Gaussia luciferase, a secreted blue luciferase with a smaller molecular weight of only 22KD, containing a natural secretory signal peptide that directs the secretion of luciferase into the cell culture supernatant. Reporter gene activity can be detected without lysing cells. More than 85% of the expressed luciferase is secreted extracellularly, but since the secreted luciferase produces signals that are many times stronger than those from Firefly or Renilla, it can also be in cells. Fluorescein remaining in the cells was detected in the lysate. Secretory expression provides great convenience for detection - real-time kinetic analysis of live cells, continuous time curve studies, without the need to lyse precious cells. It can be combined with red firefly luciferase as a two-color detection system.
Luciferase luminescence is an enzymatic reaction, and a luminescent reaction is required after the substrate is added. Secreted luciferase requires removal of the supernatant for reaction, and lytic cells are required for intracellular expression of luciferase to be detected. The advantage is that luciferase has no background in the cell, so it can detect very weak expression, with high detection sensitivity and a wide range of kinetic detection, which can be accurately quantified.
Moreover, the luciferase detection reagent can be subdivided into a flash type and a glow type according to the duration of the generated luminescent signal. The main difference between flash and glow type luminescent signals is their dynamic behavior. Upon addition of the substrate, the flash-type luminescent reaction produces a transient luminescent signal, while the glow-type luminescent reaction produces a stable luminescent signal, which may last for several hours. It is not difficult to imagine that the flash-type luminescence intensity will be larger and the detection sensitivity is higher than that of the similar glow-type enzyme. However, since the glow type detection reagent has a long duration of illumination, it is not necessary to use an autosampler, and batch processing is also easier. Special care is required when selecting test reagents.
• Simultaneous analysis of multiple regulatory elements • Simultaneous analysis of multiple signal transduction pathways • Single screening of more than one target, including off-target effects • Analysis of interactions between two or more pathways Although in the past, fluorescein was also used Enzyme combined with chloramphenicol acetyltransferase (CAT), β-galactosidase (β-Gal), or glucuronidase (GUS) as a dual reporter gene system, but luciferase quantitative detection can be in a few seconds Completed in the clock, while CAT, β-Gal and GUS require long-term pretreatment; many types of cells have endogenous β-Gal or GUS expression, which is not conducive to accurate quantitative reporter gene expression, pretreatment cell lysis at high temperature The fluid reduces the interference of endogenous β-Gal and CAT, but these treatments also rapidly inactivate luciferase. Therefore, in such dual reporter gene assays, co-transfected cell lysates must be treated separately in different steps, which is quite cumbersome.
However, the most convenient is double-secretion test: this is a two-step test that can be used to monitor Gaussia secreted luciferase and Cypridina secreted luciferase in real time. This dual-reporting system measures kinetics of luciferase from the same cell culture supernatant with high sensitivity, which not only saves sample processing time, but also reduces detection variability. The two illuminating wavelengths are close and cannot be distinguished by filters. It is necessary to divide the supernatant into two groups to detect the two enzyme activities with two substrates. Since the substrates of the two luciferases are completely different, the activity of the two enzymes can be effectively distinguished.
• Gaussia – Red Firefly luciferase • Cypridina – Red Firefly luciferase • Green Renilla – Red Firefly luciferase
Other Applications <br> Luciferase can be used as a single reporter gene to study a biological event in a specific biological experiment. Since the luminescence spectrum characteristics and substrates of different luciferases are different, it is also possible to use a plurality of different luciferases in combination for multiplex detection.
• Simultaneous study of expression regulation of multiple genes • Reduction of off-target effects • Identification of interactions between two or more signaling pathways • Normalization of “illusions†produced by experimental systems
application
Types of luciferase and application introduction
Research and exploration in the microscopic world of cells and genes, genetic reporter genes are very useful "visualization and quantification" tools with a wide range of applications. Luciferase (luciferase) is an ideal reporter gene for its excellent sensitivity, ease of use, and quantitative detection.
Luciferase is not a specific molecule. It is a general term for enzymes that catalyze the production of bioluminescence. Different sources of luciferase have their own characteristics, which can catalyze the emission of different colors of the substrate, and some can also cooperate with two-color illumination. Detection. The earliest source of this enzyme, and the most representative luciferase from the North American firefly named Photinus pyrali', is called luciferase, which also helps to distinguish the need to stimulate the light source. A fluorescent protein that excites fluorescence can be generated, and luciferase is a luminescent reaction that catalyzes the decomposition of a substrate. Luciferase has no background in the cell, so it can detect very weak expression with higher sensitivity. However, with the continuous advancement and upgrading of luminescence detection equipment, the high sensitivity of bioluminescence and the convenience of detection make these enzymes and fluorescent proteins gradually replace the trend of other reporter genes. As a result, more and more new luciferases, not from fireflies, have entered the market and are no longer the "fluorescent" world.
More types
So let's see what new luciferases are there now?
2. Cypridina luciferase is also a secreted luciferase with a beautiful blue-purple color and a protein molecular weight of 62KD. Like Gaussia, most of the luciferase products are secreted extracellularly, allowing continuous detection of live cells, which is very convenient. Due to the strong signal, residual luciferase in the cell is sufficient for routine lytic cell detection, enabling multiple assays. Cypridina luciferase can be combined with red firefly luciferase for two-color detection.
3. Red Firefly luciferase is an intracellular protein that migrates to the red spectral region compared to native Firefly luciferase (green). This is really good news for luciferase, which has always been "green fat red and thin". It is due to this spectral shift of Red Firefly luciferase that it is well distinguished from Gaussia, Cypridina or Green Renilla luciferase and can be used in combination.
4. It can share the same substrate with red and green color mites luciferase. The size is the same, the substrate is consistent, and the difference is only a few amino acids. It is a good choice for two-color detection.
5. Green Renilla (green sea kidney) luciferase is an intracellular protein with higher stability and better luminescence intensity in serum than natural Renilla luciferase. Therefore, the use of the Green Renilla luciferase reporter gene is more sensitive.
Fluorescent proteins such as GFP, which is also an ideal reporter gene, are now a colorful world. What are the characteristics of fluorescent proteins and luciferases? Fluorescent proteins are not self-luminous, but require an excitation source to generate emission fluorescence, which is derived from excitation light. The advantage of fluorescent protein is that the detection does not require substrate, non-invasive, and does not need to interfere with the cell for continuous detection; but the excitation light will cause background fluorescence interference (cells, containers, etc. will also produce certain fluorescence under the excitation light source), usually Used for qualitative or semi-quantitative.
The sensitivity of the detection reagent is higher. Nowadays, the sources of luciferase are different, each has its own advantages, and the corresponding detection reagents are naturally more and more varied and elegant. In the past, there were only firefly luciferase and Renilla luciferase, because both of them were intracellular expression, it was necessary to detect the lysis of cells first, and the firefly luciferase catalyzed the substrate to emit light for a short duration. For batch processing, it is necessary to have a test device equipped with an automatic sample introduction device, which is somewhat troublesome. Nowadays, with the secreted expression of luciferase, it is possible to directly take the supernatant for detection without lysing the cells, and it can be used for real-time kinetic analysis of living cells for continuous time curve analysis, which is much more convenient. Will the secreted luciferase enter the liquid phase and greatly reduce the signal intensity due to dilution? This does not seem to worry, the two major types of secreted luciferase-catalyzed substrate luminescence signal is 2-3 orders of magnitude higher than the original firefly luciferase and Renilla luciferase, naturally more sensitive, more suitable for micro Orifice detection.
The choice of single luciferase assay is quite rich, and researchers can freely combine different luciferases for multiple assays based on the needs of the study.
A magical new method for double report detection
When it comes to multiplex detection, it is worth noting that when luciferase is used to quantify gene expression, a second reporter gene is usually used as an internal control to homogenize the detection of the first reporter gene, thereby reducing the variation of experimental variables. For example, differences in the number and viability of cultured cells, cytotoxicity, efficiency of cell transfection and lysis.
Dual luciferase assays can be used in the following research directions:
The dual luciferase reporter gene technology, combined with firefly luciferase assay and marine coelenterazine luciferase assay, enables dual luciferase reporter gene detection in a single tube, which is fast, sensitive and simple. But the early dual luciferase technology needed to lyse the cells first, detect the firefly luciferase, then quench the firefly fluorescein while activating the renilla luciferase, and then do the second test, which seems a bit cumbersome. The Dual-Glo has been improved to become a homogeneous reagent for sample-detection.
In addition to the dual secretory assay, there are three sets of dual reporter assays (detection of intracellular luciferase):
This is a method of distinguishing the luminescence signals generated by two different luciferases in the spectral range, and double detection can be achieved in one step. By adding only one reagent (including two substrates), the two illuminating signals can be simultaneously detected by the conversion filter. In this way, the quenching step and the step-by-step detection are eliminated, and the different luciferase activities are simultaneously measured in the same sample, the operation is simple, and the result is more synchronic. The key is that the emission spectra of the two reporter genes in each combination are far enough apart to allow the instrument to be well distinguished. At this time, the significance of the spectral red shift of Red Firefly luciferase can be seen. It is worth mentioning that two secreted luciferases have been introduced. Although most of the expressed products are secreted extracellularly, there is still sufficient luciferase activity in the cells, which can be obtained by flash or glow detection reagents. Detection.
In practice, luciferase has the advantage of extremely high sensitivity and a wide dynamic range for easy quantitative analysis. The sensitivity of luciferase is highly dependent on the intensity of the fluorescence produced. In contrast, secreted luciferase, Cypridina and Gaussia luciferase have the highest luminescence intensity, followed by Renilla luciferase and finally Firefly luciferase.
Multiple detection is more suitable for the following aspects:
• Analysis of cis-acting elements and trans-acting factors in promoter studies • Drug screening • siRNA and miRNA screening • Secretory pathways and protein localization reporter assays • Real-time dynamic studies of living cells • Signal transduction pathway analysis • Difficult to transfect Research on cells (including stem cells and primary cells) • RNA splicing studies
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