Frankenbody Expression Plasmids

At a Glance

Researchers at Colorado State University have developed fluorescence microscopy tools to image proteins in living cells that are difficult if not impossible to see using standard techniques. They are using these techniques to answer long-standing questions about how epigenetic factors contribute to gene misregulation in human disease. Currently they are focusing on two hard-to-see protein populations: (1) nascent proteins in the process of being translated (while still attached to RNA) and (2) post-translationally modified proteins, particularly modified chromatin and modified transcription machinery.

These HA Frankenbody Expression Plasmids genetically encode scFvs that selectively bind the HA epitope (YPYDVPDYA) with high affinity in living cells and organisms and can be used to quantify HA-tagged protein translation, localization, and dynamics. The plasmids enable, state-of-the-art, single-molecule experiments in living cells, which we demonstrate by tracking single HA-tagged histones in U2OS cells and single mRNA translation dynamics in both U2OS cells and neurons.

Background

Antibodies are the biomolecules our immune systems deploy to find, tag and destroy invading pathogens. They work by binding to specific targets, called epitopes, on the surfaces of antigens – like locks to keys.

For many decades, scientists have cleverly exploited this selective tagging mechanism in natural antibodies to engineer antibody-based probes that let them purify and study different types of proteins within cells. One tried and true technique, epitope tagging, involves fusing an epitope to a protein of interest and using fluorescently labeled antibodies to make those proteins visible – but only in fixed, dead cells.

Researchers from Colorado State University and the Tokyo Institute of Technology have added a new tool to the arsenal of antibody-based probes, but with a powerful distinction: Their genetically encoded probe works in living cells.

Overview

HA Frankenbody Expression Plasmids

PNZ035, Pnz055 & pnz053 info/purchase

Plasmids encoding anti-HA single-chain variable fragments (scFv) fused with mEGFP, HaloTag, or mCherry under CMV promoter.

Benefits

Encode mouse anti-HA- scFvs for H. sapiens (human) expression
Engineered scFv binding the linear HA epitope with high affinity and specificity in vivo
Light up in multiple colors HA-tagged nuclear, cytoplasmic, membrane, and mitochondrial proteins in diverse cell types
Precisely quantify the expression of HA-tagged proteins in live cells
Powerful tool for imaging protein dynamics in vivo