Mass isolation of staged Drosophila pupal intestines for analysis of protein ubiquitylation

Ruoxi Wang; Panagiotis D Velentzas; Eric H Baehrecke

doi:10.1016/j.xpro.2023.102713

Mass isolation of staged Drosophila pupal intestines for analysis of protein ubiquitylation

STAR Protoc. 2023 Dec 15;4(4):102713. doi: 10.1016/j.xpro.2023.102713. Epub 2023 Nov 11.

Authors

Ruoxi Wang¹, Panagiotis D Velentzas², Eric H Baehrecke³

Affiliations

¹ Department of Molecular, Cell and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA. Electronic address: ruoxi.wang@umassmed.edu.
² Department of Molecular, Cell and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA. Electronic address: panagiotis.velentzas@umassmed.edu.
³ Department of Molecular, Cell and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA. Electronic address: eric.baehrecke@umassmed.edu.

Abstract

Large quantities of developmentally synchronized pupal intestines are required for biochemistry experiments. Here, we present a protocol for the mass isolation of staged pupal intestines during Drosophila melanogaster development based on buoyancy in sucrose for biochemical evaluation of protein ubiquitylation. We describe steps for crossing flies, preparation of samples, immunoprecipitation of proteins from staged isolated tissues, and analysis of samples by western blot. This protocol can be extended to investigate biochemical changes in other tissues. For complete details on the use and execution of this protocol, please refer to Wang et al. (2023).¹.

Keywords: Cell Biology; Developmental biology; Microscopy; Model Organisms.

MeSH terms

Animals
Blotting, Western
Drosophila melanogaster*
Drosophila*
Intestines
Pupa
Ubiquitination

Grants and funding

R35 GM131689/GM/NIGMS NIH HHS/United States