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Hassle-free 96-well format epitope tagging using Cas9 ribonucleoprotein

Posted by Guest Blogger on Jun 28, 2018 11:01:10 AM

This post was contributed by guest blogger Pooran Dewari, a postdoc in Steve Pollard’s lab at the MRC Centre for Regenerative Medicine (CRM), Edinburgh.

Most commercial antibodies do not work in pull-down assays: Epitope tagging provides a solution

Proteins - the workhorses of the cell – never work alone in the cellular milieu. It is, therefore, critical to understand how proteins interact with one another (or with DNA) to perform diverse biochemical tasks in the cell. One of the most popular approaches to study protein interactions is the pull-down assay, wherein a protein of interest can be captured along with its associated partners. Common pull-down assays include immunoprecipitation mass spectrometry (IP/MS) and chromatin immunoprecipitation (ChIP). In IP/MS, a target protein is first immunoprecipitated - along with its associated protein complexes - from the cell-lysate using antibodies against the target protein. The captured protein complexes are then analysed by mass spectrometry to identify the interacting proteins. Similarly, in ChIP-seq assays, chromatin fragments that are bound by a protein of interest are pulled-down and later coupled to high-throughput sequencing to identify genome-wide binding patterns of the target protein.

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Topics: CRISPR, Techniques

dTAG - You're it!

Posted by Guest Blogger on Jun 21, 2018 10:06:52 AM

This post was contributed by guest blogger Behnam Nabet, a postdoctoral fellow at Dana-Farber Cancer Institute.

Targeted Protein Degradation

In the Bradner and Gray labs, we synthesize compounds that enable selective removal of proteins-of-interest from the proteome. Rather than inhibiting protein function, these so-called “small molecule degraders” recruit the proteasome to destroy targeted proteins. We previously developed small molecule degraders that achieve selective degradation of endogenous proteins (notably, BRD2/3/4, CDK9, TRIM24, FLT3, BTK, and ALK) by linking small molecules that bind these target proteins to other small molecules that bind an E3 ligase. These bifunctional degraders co-opt E3 ligases such as cereblon (CRBN) or von Hippel-Lindau (VHL) to bring the endogenous degradation machinery into close proximity with the target protein, leading to polyubiquitination of the target protein and proteasomal degradation. Remarkably, small molecule degraders provide distinct advantages over pharmacological inhibitors including rapidly depleting a protein-of-interest, increasing target selectivity, overcoming resistance to inhibitors, and inducing prolonged biological effects.

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Topics: Techniques, Hot Plasmids

Pathways Over Time Plasmids Engage Students in Functional Genomics Research

Posted by Guest Blogger on May 22, 2018 9:38:25 AM

This post was contributed by guest blogger Clare O'Connor an Associate Professor at Boston College.

National reports stress the importance of providing authentic research experiences to undergraduate students (1, 2), but educators face significant challenges in designing suitable projects. In the O'Connor lab, we recognized that genome sequencing projects were generating huge amounts of data that could provide the basis for student projects in introductory labs. Genome projects use computational methods to identify genes by their similarities to genes in other species, but these studies generally leave questions about gene function wide open. What if two seemingly similar proteins have acquired divergent functions due to mutations accumulated over time? Undergraduates can help to answer this question!

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Topics: Education, Hot Plasmids

FPbase: A new community-editable fluorescent protein database

Posted by Guest Blogger on May 16, 2018 9:00:00 AM

This post was contributed by guest blogger Talley Lambert, a Research Associate at Harvard Medical School.

The need for a community fluorescent protein database

As recognized by the 2008 Nobel Prize, fluorescent proteins (FPs) have become one of the most indispensable tools in modern biological research.  Any microscopist will tell you that selection of a fluorescent probe (be it an organic dye or FP) is one of the most important steps in the design of an imaging experiment.  The choice is non-trivial, however, as FPs are tremendously complicated entities with a large range of characteristics (color, brightness, photostability, maturation, oligomerization), many of which are dramatically affected by environmental conditions (such as temperature, pH, fusion protein, etc...).  There are many online guides – including an excellent series of posts by Joachim Goedhart on the Addgene blog – outlining various important considerations when choosing a FP, but much of the primary data one might require when making such a decision remains spread across literature in publications that introduce these tools.

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Topics: Fluorescent Proteins, Scientific Sharing

Analyzing CRISPR Editing Results with ICE from Synthego

Posted by Guest Blogger on May 8, 2018 9:00:20 AM

This article was contributed by Jessica Roginsky, Scientific Support Lead at Synthego. Article source: Step-by-Step Guide for Analyzing CRISPR Editing Results with ICE on Synthego’s blog.

CRISPR-based genome engineering has revolutionized the gene editing field by making experimental workflows considerably easier, faster, and more efficient than previous methods. Still, generating reliable results from CRISPR edit data requires the help of robust software tools. As a consequence, a critical step in the gene editing workflow - analyzing the data - is often under-appreciated or over-looked. 

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Topics: CRISPR

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