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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

PEI Calculator for Planning AAV Packaging Transfections

Posted by Beth Kenkel on Jan 23, 2018 9:04:28 AM

As the saying goes, “failing to plan is planning to fail.” Preparation is a key step to any experiment and can help prevent future headaches. To help you plan PEI transfections for AAV packaging, considering using this PEI Calculator. AAV packaging typically requires transfecting three plasmids at specific molar ratios. To get these ratios right, you need to do a few calculations. This calculator can do the math for you and it’s simple to make adjustments for the type of tissue culture dish and number of dishes used. Here’s to making AAV packaging a little easier!

Click here to download the PEI calculator for AAV packaging

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Topics: Viral Vectors

Plasmids 101: Inducible Promoters

Posted by Mary Gearing on Jan 18, 2018 9:34:59 AM

Promoters control the binding of RNA polymerase and transcription factors. Since the promoter region drives transcription of a target gene, it therefore determines the timing of gene expression and largely defines the amount of recombinant protein that will be produced. Many common promoters. like CMV, EF1A, and SV40 promoters, are always active and thus referred to as constitutive promoters. Others are only active under specific circumstances. In this post, we’ll discuss inducible promoters, which can be switched from an OFF to an ON state, and how you might use these in your research. When you're done with this post, check out our follow up post on repressible promoters.

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Topics: Plasmids 101

Top Requested AAV of 2017: pmSyn1-EBFP-CRE

Posted by Tyler Ford on Jan 17, 2018 9:57:12 AM

We began distributing ready-to-use virus preps through our viral service in late 2016 and requests are still pouring in! While our lentiviral service is going strong, the AAV service has shown incredible growth this year. pAAV-hSyn-DIO-hM4D(Gi)-mCherry was the top requested AAV prep for the 2nd year running, and you can learn more about this useful, DREADD-containing AAV here. But the top requested AAV that became available in 2017 is pmSyn1-EBFP-Cre from Hongkui Zeng’s lab. This AAV has had over 150 orders since coming online!

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Topics: Plasmid Technology, Hot Plasmids, Viral Vectors

Visualizing Protein Turnover In Situ

Posted by Guest Blogger on Jan 16, 2018 10:20:10 AM

This post was contributed by guest blogger, Eugenia Rojas.

A question worthy of a PhD: How do you visualize protein turnover within a neuron?

For my PhD I studied a synaptic protein that is linked to neurodegeneration. The level of this protein is decreased in Alzheimer’s disease patient’s brains. However, it is not known why or how this happens. Therefore, I set out to study how protein turnover is regulated in neurons.

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Topics: Blog

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