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Fluorescent Biosensors for Measuring Autophagic Flux

Posted by Beth Kenkel on Jan 22, 2019 9:41:01 AM

Autophagy (Greek for “self-eating”) is a process by which cytoplasmic material, including organelles, are targeted to lysosomes for degradation. Autophagy is a dynamic process which involves autophagosome synthesis, delivery of materials to be degraded to the lysosome, and degradation of autophagic substrates inside the lysosome. Historically, methods for studying autophagy focused on counting the number of autophagosomes. This approach, however, has inherent limitations because it turns a dynamic process into a static measurement and it provides limited information about what materials or organelles are being targeted for autophagy. The development of several fluorescent autophagy reporters now allows for the measurement of autophagic flux, or the changes in autophagic activity, and are a more reliable indicator of autophagic activity. The aim of this post is to provide an overview of four autophagy biosensors currently available from Addgene.

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Topics: Fluorescent Proteins, Fluorescent Biosensors

Measuring Kinase Activity at the Single-Cell Level with Kinase Translocation Reporters (KTRs)

Posted by Beth Kenkel on Jul 26, 2018 8:46:55 AM

Kinases: they regulate many proteins, with ~1/3 of human proteins predicted to be phosphorylated on at least one site. Phosphorylation is particularly important for regulating signal transduction and measuring kinase activity at the single-cell level can aid in drawing connections between signaling activity and cell phenotype. One method for monitoring live single-cell kinase activity is FRET, but FRET reporters are challenging to design and difficult to multiplex. The Covert Lab provides an alternative tool with their Kinase Translocation Reporters (KTRs) whose cellular localization serves as a proxy measurement of kinase activity. The key advantage of KTRs is that they are easy to create and simple to multiplex.

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Topics: Fluorescent Proteins, FRET, Fluorescent Biosensors

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