Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments

Summary

The goal of this protocol is to provide a straightforward and inexpensive approach to collecting Drosophila embryos at medium scale (0.5-1 g) and preparing protein extracts that can be used in downstream proteomic applications, such as affinity purification-mass spectrometry (AP-MS).

Abstract

Analysis of protein-protein interactions (PPIs) has become an indispensable approach to study biological processes and mechanisms, such as cell signaling, organism development, and disease. It is often desirable to obtain PPI information using in vivo material, to gain the most natural and unbiased view of the interaction networks. The fruit fly Drosophila melanogaster is an excellent platform to study PPIs in vivo, and lends itself to straightforward approaches to isolating material for biochemical experiments. In particular, fruit fly embryos represent a convenient type of tissue to study PPIs, due to the ease of collecting animals at this developmental stage and the fact that the majority of proteins are expressed in embryogenesis, thus providing a relevant environment to reveal most PPIs. Here we present a protocol for collection of Drosophila embryos at medium scale (0.5-1 g), which is an ideal amount for a wide range of proteomic applications, including analysis of PPIs by affinity purification-mass spectrometry (AP-MS). We describe our designs for 1 L and 5 L cages for embryo collections that can be easily and inexpensively set up in any laboratory. We also provide a general protocol for embryo collection and protein extraction to generate lysates that can be directly used in downstream applications such as AP-MS. Our goal is to provide an accessible means for all researchers to carry out the analyses of PPIs in vivo.

Introduction

Genetic screens and, more recently, genomic approaches have revolutionized the study of biological functions. However, important cellular information is encoded in proteins and their ensemble of interacting partners. While traditional genetic modifier screens can identify rate-limiting pathway components and recover indirect interactions, the strength of the proteomic approaches lies in their ability to identify complete immediate interaction networks of proteins of interest. Proteomics is thus a valuable orthogonal method to study biological systems, and complements genomics, transcriptomics, and traditional genetic screens. Affinity purification-mass spectrometry (A....

Protocol

1. Preparation of 5 L Fly Cages (to Fit 15 cm Plates)

1. Obtain the materials needed: 5-quart (4.7-L) container with lid (Figure 1A), nylon mesh, and a razor blade.
2. Mark a 12 cm diameter circle and cut a hole in the bottom of the container using the razor blade (Figure 1B). Cut a 15 cm diameter hole in the lid (Figure 1C).
3. Cut nylon mesh into 25 x 25 cm squares.
4. Place the mesh over the top rim of the container and press down with the lid (Figure 1C). Make sure the mesh is tight and the lid is not tilted. The cag....

Results

To illustrate the use of this protocol in a protein complex purification experiment, we generated a homozygous viable fly line, arm-EGFP-ERK, expressing EGFP-tagged Drosophila extracellular signal-regulated kinase (ERK, encoded by the rolled gene) under the control of a ubiquitously expressed armadillo (arm) promoter^18,19. The arm promoter is active during most stages of Dr.......

Discussion

The protocol presented here is a simple and general procedure for setting up Drosophila population cages at medium scale and making whole-cell protein extracts from embryos. The resulting extracts can be used in a variety of downstream applications, such as purification of protein complexes on affinity resins. It is critical to perform the extraction steps on ice and use strong protease inhibition, to minimize protein degradation. As described, the protocol is suitable for isolation of most cytoplasmic and some .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Leaktite 5-Qt. Natural Multi Mix Container (Pack of 3)	Home Depot	209330	For making 5-L cages.
Leaktite 5-Qt. Natural Multi Mix Lid (Pack of 3)	Home Depot	209320	Diameter 8.37 in. Lids for 5-L cages.
Fisherbrand Petri dishes with clear lid, 100 x 15 mm	Fisher Scientific	FB0875712	Material: Polystyrene. To be used with 1-L cages.
Fisherbrand Petri dishes with clear lid, 150 x 15 mm	Fisher Scientific	FB0875714	Material: Polystyrene. To be used with 5-L cages.
Fisherbrand Petri dishes with clear lid, 60 x 15 mm	Fisher Scientific	FB0875713A	Material: Polystyrene. Used during embryo dechorionation.
Sefar NITEX nylon mesh	Sefar	03-180/44	180 micron, 44% open area, used for fly cages.
Milwaukee 3 in. Hole Dozer Hole Saw with Arbor	Home Depot	49-56-9670	3 inch cutting tool for making 1-L cages.
Nalgene Wide-Mouth Straight-Sided PMP Jars with White Polypropylene Screw Closure	Fisher Scientific	11-823-33	For making 1-L cages. Thermo Scientific cat # 21171000. Alternative 1-L containers can be used.
Red Star Active Dry Yeast, 2 pound pouch	Red Star		can be purchased from Amazon.com or other suppliers.
Frozen 100% Apple Juice Concentrate			can be purchased from a grocery store. Has to say "100% apple juice" on the can.
Methyl 4-hydroxybenzoate	Acros Organics	AC126965000	also known as methylparaben or Tegosept. Used as a preservative in AJ plates.
IGEPAL CA-630 for molecular biology, 100 ml	Sigma	I8896	used for preparing lysis buffer.
Nalgene Rapid-Flow Sterile Disposable Filter Units with CN Membrane	Fisher Scientific	09-740-2A	0.2 μm pore size. Used for filtering 5x lysis buffer. Thermo Scientific cat # 1260020.
CO-RO ROCHE cOmplete Protease Inhibitor Cocktail	Sigma	11697498001	Vial of 20 tablets. Used for protease inhibition in lysis buffer.
Corning SFCA syringe filters	Fisher Scientific	09-754-21	SFCA membrane, diameter 26 mm, pore size 0.45 μm. Used for filtering final extract samples.
BD Luer-Lok Disposable Syringes without Needles	Fisher Scientific	14-823-2A	for filtering final extract samples. BD cat # 309604.
Bleach	Clorox		used for embryo dechorionation at 50% (vol/vol) in water, can be purchased at any supermarket. It is important to use the Clorox brand, as other brands may result in incomplete dechorionation or may be toxic for embryos.
Corning Costar Netwell Plates	Fisher Scientific	07-200-213	mesh containers for embryo collections. 74 μm mesh size, 6-well.
Wheaton Dounce Tissue Grinders, capacity 15 ml	Fisher Scientific	06-435B	homogenizer for embryos, with loose and tight pestles. Wheaton cat # 357544.