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When is a Monomer not a Monomer? The Top Three Ways Your Favorite Fluorescent Protein Oligomerizes in Cells

Posted by Guest Blogger on Apr 19, 2016 10:30:00 AM

This post was contributed by guest blogger Erik L. Snapp.

Stop using EGFP/GFP for fusion proteins! Despite multiple studies in high profile journal articles, many researchers remain unaware that EGFP/GFP is prone to forming noncovalent dimers. This property of EGFP can lead to significant artifacts.

If you're using green fluorescent protein or Enhanced Green Fluorescent Protein (GFP/EGFP) for a transcriptional reporter or as a general cytoplasmic label of cells, there's no problem. You're OK. However, if you fuse your protein of interest (POI) to GFP to study the protein's behavior in cells, in solution or something in between, you are using a tag with a serious drawback. The standard EGFP plasmid that used to be sold by Clontech and is in a freezer box in just about every lab in the world, is not inert. In all seriousness, EGFP/GFP has a real nontrivial propensity to noncovalently dimerize. That means that your POI fused to GFP or another fluorescent protein (FP) could be forming dimers in cells. Why should you care? Three simple ways a dimeric FP could ruin your day (and experiment) are listed below. Solutions to avoid these all too common issues follow.

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Topics: Imaging, Fluorescent Proteins

R Bodies: Membrane-Rupturing Microscopic Tools

Posted by Guest Blogger on Apr 14, 2016 10:30:00 AM

This post was contributed by guest blogger Jessica Polka, a Postdoctoral Research Fellow with Pamela Silver. 

Most types of biological motion (whether endocytosis, vesicle trafficking, or muscle contractions) are produced by orchestrated movements of networks of proteins consuming molecular fuel sources. While the importance of understanding these complex processes can’t be overstated, we can also learn a lot from Nature’s simpler solutions to transmitting forces over long distances. For instance, how much force can be generated by conformational changes in proteins? How can information propagate through a structured material over a long distance? And can we understand such a structure well enough to engineer it to suit our purposes?

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Topics: Plasmid Technology, Synthetic Biology

Pairing CombiGEM and CRISPR for Combinatorial Genetic Screening

Posted by Guest Blogger on Apr 12, 2016 10:30:00 AM

This post was contributed by guest blogger Alan Wong.

The complexity of biological systems can hinder our attempts to study and engineer them, but what if we had a simple tool that allowed us to rapidly decode the complexity? The CombiGEM-CRISPR technology was developed with the goal of providing an easy-to-use tool to analyze the complex combinatorial genetic networks underlying your favorite biological phenotype in a scalable way. This blog post will introduce you to this new technology, and guide you through the basics of CombiGEM-CRISPR experiments.

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Topics: Plasmid Technology, Genome Engineering, CRISPR

Getting the Most from Your Lentiviral Transduction

Posted by Meghan Rego on Apr 7, 2016 10:30:00 AM

Lentiviruses are a powerful laboratory tool often employed to establish cell lines that stably express a gene of interest. While the general approach for using lentivirus, infect and select, seems simple, in actuality, many find using lentivirus to be time consuming, difficult, and lacking in reproducibility. Read on for some tips for getting the most out of your lentiviral transduction experiments.

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Topics: Lab Tips, Viral Vectors

Providing Plasmids to Researchers in Developing Countries: Addgene and Seeding Labs Team Up

Posted by Tyler Ford on Apr 5, 2016 10:30:00 AM

Researchers in developing countries often find themselves on the front lines of emerging global challenges. However, these same researchers face significant barriers to scientific discovery and career development. They are working with limited resources but limitless potential. We see it as part of our mission to enable all researchers and are proud to announce that we are teaming up with Seeding Labs to better serve those in developing countries.

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Topics: Scientific Sharing, Inside Addgene

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