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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

Plasmids 101: SunTag and Fluorescent Imaging

Posted by Mary Gearing on Mar 28, 2017 10:30:00 AM

Quick Announcement from the Plasmids 101 Team: In preparation for the release of Addgene's Fluorescent Protein eBook - our next couple of plasmids 101 posts will gain a healthy, fluorescent glow. Stay tuned for more fluorescence-based Plasmid 101 posts in the coming weeks!

In biology as in life, more is often better. More transcription factor binding sites in a promoter lead to higher transcriptional activation. Multiple nuclear localization signals (NLS) increase protein import into the nucleus. In developing their SunTag technology, the Vale and Weissman labs took this biological lesson and created a system to amplify fluorescent signals. Named for the "stellar explosion SUperNova," SunTag can help you turn up the brightness in your fluorescent imaging experiments.

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Topics: Hot Plasmids, Plasmids 101, Fluorescent Proteins

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