eGFP and other fluorescent proteins have become essential tools in many fields of biology. Several studies have used eGFP, EBFP2, and mCherry in experiments that require upregulation of the UPR (unfolded protein response) pathway. Interestingly, the eGFP mRNA sequence contains two consecutive glutamine codons.
This motif is a target for the ERN1 endonuclease. ERN1 recognizes a stem loop in EGFP mRNA and degrades it by a mechanism that requires the CAGCAG sequence.
eGFP mRNA
Green fluorescent protein (GFP) is a flexible reporter gene that may be used to visualize transcription and translation activities in vivo and in vitro. GFP is a compassionate and versatile reporter detected with a fluorescent microscope, fluorometer, or flow cytometry machine. In addition, it is readily incorporated into mammalian cells and can be expressed at high levels without any interference from endogenous proteins.
eGFP plasmid
eGFP is a green fluorescent protein (GFP) that can mark and track cells in a live cell imaging experiment. In mammalian cells, GFP exhibits strong fluorescence and has an emission peak at 509 nm. It is a commonly used reporter protein in mammalian cell culture.
GFP can also measure gene expression in individual cells using a fluorescent reporter. Gene expression variations can be found and measured using a technique known as flow cytometry. Many sectors are seeing an increase in the use of this technology, including infectious illness and cancer research.
To use this technique, the EGFP gene is inserted into a vector that contains an enhancer sequence. The eGFP mRNA sequence can be manipulated to identify specific promoters. For example, copies of the HRE (hypoxia response element) sequence can be inserted into the multiple cloning site of the pEGFP-N1 DNA vector to construct the pEGFP-HRE and pEGFP-5HRE plasmids. The resulting plasmids are then transfected into Hela cells using Lipofectamine 2000. The cells are then subjected to hypoxia or normoxia, and their eGFP fluorescence is measured by flow cytometry.
Two advantages of this system are that i) GFP is not expressed significantly in wild-type cells, and ii) the EGFP fusion protein can be used as a marker to distinguish cells from untransfected cells. This is especially important in cellular imaging experiments where cell morphology and dynamics are critical.
eGFP recombinant protein
Using eGFP in cell labeling and live cell imaging allows researchers to monitor the dynamics of specific types of cells. For example, eGFP can be used to track the movements of AMPA receptors on neuronal membranes. This information can be used to understand the function of brain circuitry.
The native GFP structure contains a p-hydroxybenzylidene-imidazolidone chromophore that emits green light at 508 nm. The chromophore is generated by an internal autocatalytic reaction involving three residues on the interior alpha helix of GFP. This mechanism is susceptible to changes in pH, making it an ideal reporter for analyzing oxidative stress.
Researchers can alter the fluorescence of GFP by mutating its primary sequence. Alternatively, they can also omit one of its secondary structural elements. This can be done by truncating the circular permutant. The forgotten section is usually non-fluorescent but sometimes produces fluorescent protein when co-expressed with the entire sequence. This technique is called Leave-One-Out (LOO).
LOO GFPs can be programmed to accept any desired protein as a binding partner and to switch on fluorescence only when the target complex binds. This can be done using computational design and high-throughput screening. This technology could replace antibody-based assays in cellular imaging and other biomedical applications. However, researchers should carefully validate any experiments that use LOO-GFP with alternative methods.
eGFP fusion protein
Green fluorescent protein (GFP) is a valuable tool for scientists to visualize cell and tissue dynamics. GFP fusion proteins can be used to tag specific protein targets and can be transiently or stably expressed. These tagged proteins can be detected by fluorescence imaging. They are also helpful as markers for cell fractionation, protein localization and as a reporter of expression level.
In the early 1990s, GFP and its separate luminescent partner aequorin were first purified from the jellyfish Aequorea victoria. Aequorin reacts with Ca2+ ions to produce a blue glow, and GFP is a bright green color that binds to the aequorin binding domain. GFP was subsequently cloned and sequenced by the Prasher lab, and two amino acid mutations were made to enhance brightness and protein folding (S65T and P64L).
One of the most common applications for FFP is tagging other proteins with them to create a fluorescent molecular beacon. These chimeras enable the host protein to perform its normal function, while the piggyback fluorescent protein reports its location in the cell.
However, several issues can arise when using fusion proteins. For example, EGFP is prone to dimerization, which can cause artifacts in some experiments. To prevent this, researchers should use a monomeric FP (usually denoted by an “m” before the gene name, such as mCherry) whenever possible.
