U of Dundee Hay Lab - NEDP1_ARMeD_knockdown

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ARMeD_NEDP1_2019-11-02_13-41-15.sky.zip2020-05-25 20:17:53751481622,58918
Antibody RING-Mediated Destruction (ARMeD) of endogenous proteins

  • Organism: Homo sapiens
  • Instrument: Q Exactive
  • SpikeIn: No
  • Keywords: ARMeD Endogenous Protein Knockdown
Abstract
To help understand the cellular function of a gene the encoding DNA or mRNA transcript can be manipulated and the consequences observed. However, these approaches do not have a direct effect on the protein product of the gene, which is either permanently abrogated or depleted at a rate defined by the half-life of the protein. Therefore, complete gene deletions and RNA interference of proteins with a very long half-life risk obscuring the direct consequences via compensatory or secondary cellular responses. We therefore sought to develop a single-component system that could induce the rapid degradation of the specific endogenous protein itself. A genetically introduced inducible construct of the RING domain of the ubiquitin E3 ligase RNF4 with a protein-specific camelid nanobody mediates destruction of the target by the ubiquitin proteasome system, a process we describe as Antibody RING-Mediated Destruction (ARMeD). The technique is acutely specific as we observed no off-target protein destruction. Furthermore, bacterially produced nanobody-RING fusion proteins electroporated into cells induce degradation of target within minutes. With the increasing availability of protein-specific nanobodies this method will allow the rapid and specific degradation of a wide range of endogenous proteins.
Experiment Description
As part of a larger project involving ARMeD targeted destruction of proteins, knock down NEDP1 in HeLa cultures by doxycycline induction of anti-NEDP1-nanobody-RING-domain fusion protein and quantify the fold-reduction using PRM mass spectrometry.
Sample Description
Trypsin digested protein from HeLa whole cell extracts from WT, RING-anti-NEDP1-nanobody, and GFP-nanobody-RING strains, with or without doxycycline, in biological triplicate.
Created on 5/25/20, 8:18 PM