Question

Write a one-page summary (12-pt Arial, single line spacing) on the working mechanism of the fluorescent proteins and how fluorescent proteins can be visualized through specific fluorescent filter sets.

With work sited please!

Answer 1

Fluorescent proteins :-

  The wide spectrum of fluorescent proteins and derivatives uncovered thus far are quite versatile and have been successfully employed in almost every biological discipline from microbiology to systems physiology. These unique probes have proven extremely useful as reporters for gene expression studies in both cultured cells and entire animals. In living cells, fluorescent proteins are most commonly utilized to track the localization and dynamics of proteins, organelles, and other cellular compartments, as well as a tracer of intracellular protein trafficking. Quantitative imaging of fluorescent proteins is readily accomplished with a variety of techniques, including widefield, confocal, and multiphoton microscopy, to provide a unique window for exposing the intricacies of cellular structure and function.

The complex spectral and physical properties of fluorescent proteins affect the accuracy and utility of any quantitative measurement. Many of these properties, such as the molar extinction coefficient, quantum yield, photobleaching rate, and pH dependence on spectral profiles, can be easily measured with purified proteins in vitro. However, other important and experimentally critical properties, including the time course of chromophore formation (maturation) and protein degradation rates in vivo, are more difficult to ascertain. For typical experiments, the brightest, most stable monomeric fluorescent protein derivative should be selected to reduce the necessity for applying complicated background and photobleaching corrections in less demanding applications.

In selecting fluorescent protein vectors for imaging experiments, the commercially available enhanced variants from jellyfish and reef corals, as well as their derivatives should be seriously considered. These are available with fluorescence profiles in the cyan (ECFP), green (EGFP), and yellow (EYFP) spectral regions, and new monomeric red-emitting fluorescent proteins have now been introduced. The fluorescence emission spectral profiles for the probes depicted cover a bandwidth of almost 180 nanometers, featuring maxima that range from 440 to 618 nanometers. These probes are the brightest, most stable fluorescent protein variants and permit imaging with illumination at low light levels, which minimizes problems such as photobleaching (discussed below) and phototoxicity. Many of the fluorescent proteins variants (including EGFP, ECFP, and EYFP) can form dimers at sufficiently high concentrations, an artifact that can perturb membrane structure or lead to incorrect assumptions when conducting quantitative advanced fluorescence techniques, such as resonance energy transfer (FRET).

In addition to the intrinsic brightness of genetically engineered fluorescent proteins, the expression level is another factor to consider in producing a significantly bright intracellular signal from a fusion construct. Use of vectors with strong promoters to enhance transcription levels and the application of proper codons to optimize translation are essential for increasing expression levels of the fusion protein, thereby enhancing the overall signal level. This is particularly important when cellular autofluorescence makes it difficult to distinguish fluorescent fusion protein emission from background fluorescence.

A variety of techniques can be applied to enhance the expression level and brightness of cells harboring a fluorescent protein fusion product. Addition of sodium butyrate (approximately 1 to 5 millimolar) to the culture medium will increase the overall gene expression levels in stable cells lines expressing a fusion protein. In addition, the use of transiently transfected cells is also an attractive option, because they often display much higher levels of expression than stably transfected cells. A degree of caution should be exercised with transient transfections, however, due to possible over expression artifacts, including protein aggregation or saturation of protein targeting machinery, leading to inappropriate localization. Finally, increasing the number of tandem fluorescent protein sequences (double or triple) in the cloning vector is an alternative method for increasing fusion protein brightness levels.