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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

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

Top Requested Plasmid of 2017 - pMD2.G

Posted by Tyler Ford on Jan 10, 2018 11:13:59 AM

Plasmid technologies are constantly evolving, but sometimes a technology is so useful it forever enhances biological research and discovery. CRISPR is a great example (the top requested plasmids from 2015 and 2016 were CRISPR plasmids), but so are lentiviral vectors, many of which are used to deliver Cas9 and other genes to mammalian cells. For this reason, the top requested plasmid of 2017 is the lentivirus envelope plasmid pMD2.G from Didier Trono’s lab!

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Topics: Plasmid Technology, Hot Plasmids, Viral Vectors

15 Hot Plasmids from 2017

Posted by Tyler Ford on Jan 9, 2018 10:02:27 AM

Every quarter we highlight a subset of the new plasmids in the repository through our hot plasmids articles. These brief articles provide a synopsis of a plasmid or group of plasmids' functions and applications. We hope that these articles make it easier for you to find and use the plasmids you need. You can find all the hot plasmids from 2017 below. With over 50,000 plasmids, we can't write posts for every great plasmid that comes into the repository, but be sure to let us know if you'd like to write about your plasmids in a future blog post. No time to read?

Listen to our hot plasmids segment on the Addgene Podcast.

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Topics: Hot Plasmids, News

pCXLE toolkit: Efficient episomal plasmid-based method to reprogram peripheral blood cells to iPSCs

Posted by Guest Blogger on Dec 14, 2017 9:08:09 AM

This post was contributed by Kusumika (Kushi) Mukherjee, a Postdoc at Massachussetts General Hospital.

A little over a decade back when Yamanaka and colleagues reported that it is possible to reprogram differentiated cells into induced pluripotent stem cells (iPSCs) by the addition of reprogramming (or “Yamanaka”) factors, they changed the landscape of regenerative medicine. Their work opened up vast possibilities for the clinical and therapeutic applications of iPSCs. The generation of human iPSCs (hiPSCs) now provides an opportunity to develop and use patient-specific somatic cells that are otherwise difficult to obtain. These can then be used to perform cell therapy and to model diseases in vitro.

Find stem cell plasmids at Addgene

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Topics: Plasmid Technology, Hot Plasmids, Stem Cells

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