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Overcoming the challenges of lentiviral production

Posted by Meghan Rego on Aug 7, 2018 8:31:12 AM

While lentiviral vectors are popular gene delivery tools, producing lentivirus, can pose certain challenges. Whether choosing a system that is the best fit for the experiment, trying to produce virus of a usable titer, or fine-tuning selection and expression in your target cell line, researchers often find themselves faced with a roadblock. In this post, we will provide an overview of some of the common challenges associated with producing and using lentivirus and offer some tips and tricks for overcoming these hurdles.

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Topics: Viral Vectors, Techniques, Lab Tips

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

CUT&RUN: An Improved Method for Studying Protein-DNA Interactions

Posted by Guest Blogger on Feb 13, 2018 9:51:55 AM

This post was contributed by guest blogger Matthew J. Niederhuber, a graduate student at UNC Chapel Hill.

Chromatin immunoprecipitation followed by high-throughput sequencing, ChIP-Seq, is the go-to method for mapping where a protein binds genome-wide, and has been widely applied in many model organisms and cell lines. Although ChIP-seq is a relatively simple and robust protocol it does have limitations. The enzyme-based CUT&RUN method overcomes many of these limitations and makes it easier for you to map protein-DNA interaction with limited biological materials.

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Topics: Plasmid Technology, Techniques

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: Hot Plasmids, Plasmid Technology, Techniques

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