Plasmids 101: Fluorescent Biosensors

Posted by Jessica Welch on May 18, 2017 10:30:00 AM

Addgenie Mary Gearing contributed to the content of this article.

Biosensors (‘biological sensors’) are biological tools that monitor a process or detect a given molecule. The sensor component is usually a protein that undergoes a conformational change in response to the molecule it detects. This change then generates a reporter signal. Reporter signals may be electrochemical or light-based, with luminescent and fluorescent reporters being especially popular. We’ll give you an introduction to fluorescent biosensors, but keep in mind that there is a lot of variety in how biosensors work, and you should always check the associated publication for the specifics of your chosen plasmid.

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Topics: Fluorescent Proteins

Plasmids 101: Fluorescent Protein Timers

Posted by Tyler Ford on May 4, 2017 10:30:00 AM

Even before fluorescent proteins (FPs) came into wide use, there were a variety of ways to monitor cell, organelle, and protein localization. For instance, you might dye your cells and look at them under a microscope, fractionate samples to isolate particular organelles and their contents, or perform in situ hybridization experiments. In many cases fluorescent proteins have usurped old methods or complemented them in ways that make them much easier. A special class of FPs, the FP timers, add an entire new dimension to monitoring localization; using FP timers, researchers can look at a single image of a cell and understand how protein localization changes over time.
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Topics: Plasmids 101, Fluorescent Proteins

Plasmids 101: Photoactivatable Fluorescent Proteins

Posted by Michelle Cronin on Apr 25, 2017 10:30:00 AM

Fluorescent proteins (FPs) offer scientists a simple yet powerful way to tag cellular proteins and investigate protein localization, interaction, and expression.  However, one caveat of FP-protein fusions (FP-chimeras) is that they undergo normal protein turnover. FP-chimeras are continuously synthesized and degraded within the cell, so at any given time, an FP-chimeric protein may be at any one of many stages of synthesis and degradation. For this reason it is virtually impossible to determine specific protein turnover or temporal expression using standard FP-chimeric proteins.

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Topics: Plasmids 101, Fluorescent Proteins

Rosella: A Fluorescent pH-Biosensor for Studying Autophagy

Posted by Beth Kenkel on Apr 13, 2017 10:30:00 AM

Rosella is a pH-sensitive fluorescent biosensor that was recently deposited with Addgene by Dr. Mark Prescott. This system was developed for monitoring and analyzing autophagy of cytosol and organelles in yeast cells. Autophagy (Greek for “self-eating”) is induced by a lack of nutrients and targets cytosol and organelles to the vacuole/lysosome for degradation and recycling. The key to Rosella’s autophagy-sensing abilities is that its fluorescence emission spectra changes when it goes from a more neutral pH compartment, ­­like the cytosol, to the higher pH of the vacuole. Read on to learn more about prior methods for studying autophagy and how Rosella improves upon them.

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Topics: Fluorescent Proteins

Plasmids 101: Aptamer Fluorophores

Posted by Eric J. Perkins on Apr 11, 2017 10:30:00 AM

What is an Aptamer?

Nearly 30 years ago, two independent groups, led by Jack Szostak and Larry Gold, developed methods for selecting and amplifying RNA sequences that could bind very specifically to target molecules. Using a technique called systematic evolution of ligands by exponential enrichment (SELEX), some 1010 oligonucleotides could be screened for their affinity to a wide range of non-nucleotide targets. These RNA molecules, which could bind their targets with high specificity and affinity, were eventually called aptamers, from the Latin aptus, meaning “to fit”. SELEX could be used to classify DNA aptamers as well, and over the course of the next two decades, these nucleotide-based ligand binders would prove to be highly adaptable tools.

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Topics: Plasmids 101, Fluorescent Proteins

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