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Hot Plasmids April 2018 - Protein Degradation, Nanoscopy, FIRE-Cas9, and Yeast Expression Tools

Posted by Various Addgenies on Apr 23, 2018 10:00:31 AM

Every quarter we highlight a subset of the new plasmids in the repository through our hot plasmids articles. These articles provide brief summaries of recent plasmid deposits and we they'll make it easier for you to find and use the plasmids you need. Below you'll find our hot plasmid articles from the first quarter of 2018. If you'd ever like to write about a recent plasmid deposit please sign up here.

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TRIM-Away: Targeted Endogenous Protein Degradation

Article contributed by Mary Gearing

CRISPR and RNAi have helped researchers alter DNA sequence and RNA expression - but what if you just want to change protein levels? Until recently, no such tool existed for rapid, customizable protein degradation. Enter TRIM-Away - an antibody-based system developed by the James and Schuh labs. Clift et al. applied TRIM-away to nine proteins in ten ddifferent cell types, including difficult-to-manipulate primary cells, achieving knockdown within 1-2 hr of reagent delivery. TRIM-Away is suitable for both short-and long-lived proteins, and it can even distinguish between protein variants when used with a variant-specific antibody.

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Topics: Hot Plasmids

AAVs for Genome Editing

Posted by Tyler Ford on Mar 27, 2018 9:32:43 AM

Guest blogger Todd Waldman, Professor at Georgetown University, contributed to this post.

Adeno-associated viruses (AAVs) make fantastic gene delivery vehicles for episomal gene expression and are particularly useful for gene delivery to the nervous system. For many years they have also been used to enhance the efficiency of genome editing. In this post we'll walk through a variety of ways you can use AAVs to improve your genome editing experiments (with and without targeted nucleases).

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Topics: Hot Plasmids, Viral Vectors

New Neuroscience Tool: The SF-iGluSnFr Glutamate Sensor

Posted by Tyler Ford on Mar 8, 2018 9:00:00 AM

In a previous blog post we discussed how fluorescent proteins can be used to construct biosensors, biological tools that monitor processes or detect molecules. Here we’ll be diving into the details surrounding SF-iGluSnFr, a recently upgraded biosensor designed to detect glutamate.

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Topics: Hot Plasmids, Fluorescent Proteins

Plasmids 101: Secondary Nanobody Toolbox

Posted by Beth Kenkel on Feb 27, 2018 9:04:41 AM

Western blots. ELISAs. Immunofluorescence. What do all of these techniques have in common? They all typically require secondary antibodies, frequently of the mouse or rabbit variety. While antibodies certainly aren’t “broken,” their production does require continued animal sacrifice. Could there be an alternative method for immunodetection? Enter the Görlich lab and their anti-mouse and -rabbit IgG secondary nanobodies toolbox. Nanobodies are like tiny antibodies which work just as well, if not better, than antibodies for all of the above listed molecular techniques, but they can also be expressed in bacteria and extracted with common protein purification methods. Read on to learn more about nanobodies and how their structure and function compare to IgG antibodies, as well as how to produce them for use in your lab.

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Topics: Hot Plasmids, Plasmids 101

In Vivo Biotinylation of Bacterial Fusion Proteins

Posted by Guest Blogger on Jan 25, 2018 9:09:35 AM

This post was contributed by guest blogger Jon Backstrom, a biochemist in the Vanderbilt Eye Institute and Tonia Rex's lab.

A common strategy to determine the binding kinetics of a purified protein involves immobilization on a solid support. This allows washing away of unbound material to calculate the amount of bound ligand (after subtracting out non-specific binding). Historically, glutathione-S-transferase (GST) fusion proteins have been immobilized on a reduced glutathione matrix. The advantage of a fusion protein is the efficient purification of an already immobilized target protein. The disadvantage is that the GST moiety, which forms dimers, may influence binding kinetics of the target ligand. Another important consideration is whether the affinity of an experimental protein-ligand interaction approaches that of GST-glutathione.

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Topics: Plasmid Technology, Hot Plasmids, Techniques

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