Using Phosphoserine to Study Protein Phosphorylation

Posted by Guest Blogger on Jun 23, 2016 10:30:00 AM

This post was contributed by guest blogger Natalie Niemi, a postdoctoral fellow at the Morgridge Institute for Research in Madison, Wisconsin.

It is commonly cited that approximately one-third of cellular proteins are modified through phosphorylation (1). However, the expansion of studies on protein phosphorylation in an array of model systems coupled with advances in mass spectrometry suggest that phosphorylation is far more prevalent than previously appreciated. PhosphoSitePlus, one of the most inclusive databases of post-translational modifications, identifies a staggering ~250,000 phosphorylation events in the proteomes of higher mammals (2). How can we begin to understand the importance of any of these phosphorylation events on the activity of a given protein?

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Topics: Plasmid How To, Synthetic Biology, Lab Tips, Techniques

Evolution of Lab Techniques

Posted by Guest Blogger on Jun 21, 2016 10:30:00 AM

This post was contributed by guest blogger, Krissy Lyon, a PhD candidate in Neuroscience at Harvard University.

Just as computers, cell phones, and cars become more technologically advanced leaving earlier versions obsolete, the techniques we use in lab are replaced by improved versions that save both time and money. Yet, knowledge of historical techniques comes in handy whether you are perusing classic papers or are brainstorming new technological innovations. Let’s take a look at three historical techniques: southern blotting, restriction mapping, and sequencing gels, as well as their modern equivalents and see what we can learn from their evolution.

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Topics: Fun, Lab Tips, Techniques

Genome engineering using Cas9/gRNA Ribonucleoproteins (RNPs)

Posted by Joel McDade on Apr 21, 2016 10:30:00 AM

CRISPR has quickly become the preferred system for genome engineering due to its simplicity, as it requires only Cas9 and a guide RNA (gRNA).  Choosing the correct method to deliver both Cas9 and gRNAs to your target cells is absolutely critical as failure to adequately express either component will result in a failed experiment.  In our previous blog post entitled “CRISPR 101 - Mammalian Expression Systems and Delivery Methods” we provided a general overview of the most common ways in which you can deliver Cas9 and gRNAs to your target cells and discussed a few key advantages and disadvantages of each method. In this blog post, we will go into greater detail about why and how Cas9/gRNA Ribonucleoprotein complexes (Cas9 RNPs) are being used for genome engineering experiments and provide a general framework for getting started with Cas9 RNPs in your research.

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Topics: Genome Engineering, CRISPR, Techniques

Tips for Titering Your Lentiviral Preps

Posted by Meghan Rego on Mar 15, 2016 10:30:00 AM


The day has arrived; you’ve painstakingly cared for your packaging cell line, prepped your DNA, transfected and harvested your lentivirus. Now it’s time to move ahead with your infection and make your stable cell line. While we’ve all experienced the pressure to move a project forward, transductions should not be rushed into. Before you start any transduction, you should always titer your virus - that is determine the amount of virus you actually have in your prep. Taking time to properly titer your virus will not only ensure that your infection is designed in the best possible way but it may also save you time in the long run. Read on for an overview of the titering options and the benefits and drawbacks of different methods (for comprehensive protocols for all of the methods discussed here refer to
Kutner et. al.).

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Topics: Techniques, Viral Vectors

PITChing MMEJ as an Alternative Route for Gene Editing

Posted by Mary Gearing on Feb 23, 2016 10:30:00 AM

If you follow CRISPR research, you know all about using non-homologous end-joining (NHEJ) to make deletions or homology-directed repair (HDR) to create precise genome edits. But have you heard of another double-stranded break repair mechanism: MMEJ (microhomology-mediated end-joining)? MMEJ, a form of alternative end-joining, requires only very small homology regions (5-25 bp) for repair, making it easier to construct targeting vectors. Addgene depositor Takashi Yamamoto’s lab has harnessed MMEJ to create a new method for CRISPR gene knock-in, termed PITCh (Precise Integration into Target Chromosomes). Using their PITCh plasmids, GFP knock-in cell lines can be created in about a month and a half, without the need for complicated cloning of homology arms.

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Topics: Plasmid Technology, Genome Engineering, CRISPR, Techniques

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