The reporter gene and the foreign gene are placed in the same DNA construct and then inserted in the cell. Here, the reporter gene is used as a selectable marker to find out the successful uptake of foreign genes.

In molecular biology, a reporter gene (often simply reporter) is a gene that researchers attach to a regulatory sequence of another gene of interest in bacteria, cell culture, animals or plants.

In eukaryotes, gene fusions use different reporter genes. For example, yeast reporter genes include CUP1, a gene that enables yeast to grow on copper-containing media, URA3, a gene that kills yeast when growing on 5-fluorouracil, and ADE1 and ADE2, two genes that synthesize adenine.

Luciferase-based assays are better than other reports because of the following advantages: Quick and real-time measurement. Exceptionally high sensitivity than fluorescent reporters like GFP (10- to 1,000 fold) Range of measurement is wide and dynamic.

Since the cloning and enhancement of the green fluorescent protein (GFP) derived from the jellyfish Aequorea victoria (4, 7, 9, 27–29, 41, 46), GFP has been widely used as a reporter gene.

In biological research, luciferase is commonly used as a reporter to assess the transcriptional activity in cells that are transfected with a genetic construct containing the luciferase gene under the control of a promoter of interest.

The primary advantages of either luciferase over the other reporter genes described above are their combination of high specific activity and low background (mammalian cells do not express light-generating enzymes), large and linear dynamic range (7–8 orders of magnitude), and the relatively rapid turnover of the …

What are the disadvantages of ATP bioluminescence assay?

• It does not easily distinguish ATP from microorganisms, animals, and plants.
• Luminescence from food can affect the actual ATP bioluminescence readings.
• The presence of detergents, sanitizers, or other chemicals also can affect the readings.

SDS- PAGE analysis showed that the major protein expressed had a molecular weight similar to authentic luciferase. Flow cytometry and insect luciferases with clearly separated emission spectra appear to be of value for sensitive in vivo analysis of gene promoter activity.

I used GFP expression in a dual-reporter study and its expression did not influence the luciferase activity in any way, nor do I think it should. Although the emission spectrum for luminescence is often similar to that for fluorescence, the manner in which the emission is achieved differs.

Two types of luciferase protein are commonly used, firefly (Photinus pyralis) and bacterial. Firefly luciferase uses luciferin as a substrate, oxidizing it to oxyluciferin in a reaction that utilizes molecular oxygen and ATP, and liberates light at 560 nm (Wilson and Hastings, 1998; Fraga, 2008).

Many luciferase assays require cell lysis as the most efficient means to disrupt the cell membrane and deliver the substrate; however, secreted luciferase or the use of alternative substrate reagents can permit measurements of luminescence from live cells.

GFP can be excited by the 488 nm laser line and is optimally detected at 510 nm.

Flow cytometry and fluorescent microscopy are two conventional tools to detect the GFP signal; flow cytometry is an effective and sensitive technique to quantitatively analyze fluorescent intensity, while fluorescent microscopy can visualize the subcellular location and expression of GFP.

The main difference between GFP and EGFP is that the GFP (stands for Green Fluorescent Protein) is a protein that exhibits bright green fluorescence when exposed to blue light whereas the EGFP (stands for Enhanced Green Fluorescence Protein) exhibits stronger fluorescence than GFP.

Green fluorescent protein (GFP) can be directly visualized in living cells, tissues or organisms under UV illumination. This advantage of GFP is exploited in the development of a practical approach in which GFP is used as a visual marker to monitor the removal of the selectable marker gene from transgenic plants.

Biologists use GFP to study cells in embryos and fetuses during developmental processes. Biologists use GFP as a marker protein. GFP can attach to and mark another protein with fluorescence, enabling scientists to see the presence of the particular protein in an organic structure.

Assessing Expression through Microscopy Looking for fluorescence through microscopy is one of the ways through which GFP fusion expression can be assessed. This approach has been successfully used to show that a GFP moiety has been accurately expressed making a straightforward method of assessing fusion expression.

GFP fusions permit analysis of proteins in living cells and offer distinct advantages over conventional immunofluorescence. Among these are lower background, higher resolution, robust dual color colocalization, and avoidance of fixation artifacts.

Immunofluorescence is only limited to fixed (i.e., dead) cells when structures within the cell are to be visualized because antibodies do not penetrate the cell membrane when reacting with fluorescent labels. Use of such “tagged” proteins allows determination of their localization in live cells.

Immunofluorescence allows researchers to evaluate whether or not cells or tissues in a particular sample express the antigen in question. In cases where an immunopositive signal is found, immunofluorescence also allows researchers to determine which subcellular compartments are expressing the antigen.

In clinical immunodermatology, there are three basic types of immunofluorescence techniques: direct immunofluorescence (DIF), indirect immunofluorescence (IIF) [Figure 1], and complement binding indirect immunofluorescence.

Immunofluorescence is an assay which is used primarily on biological samples and is classically defined as a procedure to detect antigens in cellular contexts using antibodies. The specificity of antibodies to their antigen is the base for immunofluorescence.

Direct immunofluorescence involves the exclusive use of antibodies which have been covalently coupled with fluorochromes. The specimen is incubated with the labelled antibody, unbound antibody is removed by washing, and the specimen is examined.

Direct immunofluorescence technique: it is a one-step histological staining procedure for identifying in vivo antibodies that are bound to tissue antigens, using a single antibody labeled with a fluorophore [5] for staining the tissues or cells. The antibody recognizes the target molecule and binds to it.

Immunofluorescence is a technique that is used to detect the presence of specific antigens in a sample. Since several secondary antibodies can bind with a primary antibody and several fluorophores can conjugate with secondary antibodies, indirect immunofluorescence is a more sensitive method than the direct method.

Primary antibodies bind to the antigen detected, whereas secondary antibodies bind to primary antibodies, usually their Fc domain. Secondly, primary antibodies are always needed in immunoassays, whereas secondary antibodies are not necessarily needed, which depends on experimental method (direct or indirect labeling).