Question

How to design an experiment to investigate the expression of three different markers at once using three different fluorescent stains? Please explain your rationale in choosing these stains.

Answer 1

Answer: Fluorescent markers are used to investigate proteins in a different biological environment. These fluorescent stains have the ability to absorb light and emit it at a well-defined wavelength.

Multiple straining of antigens is done in order to obtain a direct overview of multiple antigen localizations in a single tissue or in the cellular sample or to test the colocalization in the sample. These fluorescent probes are employed in general to detect proteins location and its activation and to identify protein complex formation and the conformational changes and to monitor the ongoing biological processes.

The rationale for choosing stains:

Fluorescence molecules such as FITC (fluorescein isothiocyanate) are generally used to stain cells which provides the ability to examine these cells under a fluorescence microscope. Now, we see that Multiple fluorescent markers are used to stain different parts of a cell. The Fluorescence microscopy helps in determining the exact location of specific proteins in a cell. One can even track the movement of these proteins and derive possible functions for these proteins of interest.

fluorophore we choose depends on the experiment we are doing. A fluorophore dashboard allows us to clearly visualize the performance of a selected fluorophore and in order to determine whether it is suitable for our application and where it can be used to best effect

For example,

Green Fluorescent Protein (GFP) is a fluorescent protein which is frequently used as a marker to monitor gene expression and cell division.

Fluorescent Sensors for Nitric Oxide is used as a method for detecting the presence of NO or nitric oxide in a system, Nitric oxide is a prominent compound which is widely associated with signaling pathways in various living organisms.