Name: medium-scale preparation of drosophila embryo extracts for proteomic experiments
Uploaded: 2017-05-30T15:00:00
Description: Watch this Scientific Journal Video about Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments at JoVE.com

The authors thank members of the Veraksa laboratory for helpful comments on the manuscript and suggestions for protocol improvements. A.V. was supported by the NIH grants GM105813 and NS096402. L.Y. was supported by the University of Massachusetts Boston Sanofi Genzyme Doctoral Fellowship.

1. Preparation of 5 L Fly Cages (to Fit 15 cm Plates)
<ol>
	<li>Obtain the materials needed: 5-quart (4.7-L) container with lid (Figure 1A), nylon mesh, and a razor blade.</li>
	<li>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).</li>
	<li>Cut nylon mesh into 25 x 25 cm squares.</li>
	<li>Place the mesh over the top rim ...

<ol>
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N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+during+the+life+cycle+of+Drosophila+melanogaster.">Gene expression during the life cycle of Drosophila melanogaster.</a> Science. 297 (5590), 2270-2275 (2002).</li><li>Soeller, W. C., Poole, S. J., Kornberg, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+transcription+of+the+Drosophila+engrailed+gene.">In vitro transcription of the Drosophila engrailed gene.</a> Genes Dev. 2 (1), 68-81 (1988).</li><li>Kamakaka, R. T., Kadonaga, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+soluble+nuclear+fraction,+a+highly+efficient+transcription+extract+from+Drosophila+embryos.">The soluble nuclear fraction, a highly efficient transcription extract from Drosophila embryos.</a> Methods Cell Biol. 44, 225-235 (1994).</li><li>Kyriakakis, P., Tipping, M., Abed, L., Veraksa, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tandem+affinity+purification+in+Drosophila:+The+advantages+of+the+GS-TAP+system.">Tandem affinity purification in Drosophila: The advantages of the GS-TAP system.</a> Fly (Austin). 2 (4), 229-235 (2008).</li><li>Shaffer, C. D., Wuller, J. M., Elgin, S. 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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stringent+analysis+of+gene+function+and+protein-protein+interactions+using+fluorescently+tagged+genes.">Stringent analysis of gene function and protein-protein interactions using fluorescently tagged genes.</a> Genetics. 190 (3), 931-940 (2012).</li><li>Veraksa, A., Bauer, A., Artavanis-Tsakonas, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+protein+complexes+in+Drosophila+with+tandem+affinity+purification-mass+spectrometry.">Analyzing protein complexes in Drosophila with tandem affinity purification-mass spectrometry.</a> Dev Dyn. 232 (3), 827-834 (2005).</li><li>Mukherjee, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+Notch+signalling+by+non-visual+beta-arrestin.">Regulation of Notch signalling by non-visual beta-arrestin.</a> Nat Cell Biol. 7 (12), 1191-1201 (2005).</li><li>Gilbert, M. M., Tipping, M., Veraksa, A., Moberg, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Screen+for+Conditional+Growth+Suppressor+Genes+Identifies+the+Drosophila+Homolog+of+HD-PTP+as+a+Regulator+of+the+Oncoprotein+Yorkie.">A Screen for Conditional Growth Suppressor Genes Identifies the Drosophila Homolog of HD-PTP as a Regulator of the Oncoprotein Yorkie.</a> Dev Cell. 20 (5), 700-712 (2011).</li><li>Anjum, S. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+Toll+Signaling+and+Inflammation+by+beta-Arrestin+and+the+SUMO+Protease+Ulp1.">Regulation of Toll Signaling and Inflammation by beta-Arrestin and the SUMO Protease Ulp1.</a> Genetics. 195 (4), 1307-1317 (2013).</li><li>Degoutin, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Riquiqui+and+minibrain+are+regulators+of+the+hippo+pathway+downstream+of+Dachsous.">Riquiqui and minibrain are regulators of the hippo pathway downstream of Dachsous.</a> Nat Cell Biol. 15 (10), 1176-1185 (2013).</li><li>Zhang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ecdysone+receptor+coactivator+Taiman+links+Yorkie+to+transcriptional+control+of+germline+stem+cell+factors+in+somatic+tissue.">The ecdysone receptor coactivator Taiman links Yorkie to transcriptional control of germline stem cell factors in somatic tissue.</a> Dev Cell. 34 (2), 168-180 (2015).</li><li>Lemmon, M. A., Schlessinger, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+signaling+by+receptor+tyrosine+kinases.">Cell signaling by receptor tyrosine kinases.</a> Cell. 141 (7), 1117-1134 (2010).</li><li>Vincent, J. P., Girdham, C. H., O'Farrell, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cell-autonomous,+ubiquitous+marker+for+the+analysis+of+Drosophila+genetic+mosaics.">A cell-autonomous, ubiquitous marker for the analysis of Drosophila genetic mosaics.</a> Dev Biol. 164 (1), 328-331 (1994).</li><li>Zhai, B., Villen, J., Beausoleil, S. A., Mintseris, J., Gygi, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphoproteome+analysis+of+Drosophila+melanogaster+embryos.">Phosphoproteome analysis of Drosophila melanogaster embryos.</a> J Proteome Res. 7 (4), 1675-1682 (2008).</li><li>Franco, M., Seyfried, N. T., Brand, A. H., Peng, J., Mayor, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+strategy+to+isolate+ubiquitin+conjugates+reveals+wide+role+for+ubiquitination+during+neural+development.">A novel strategy to isolate ubiquitin conjugates reveals wide role for ubiquitination during neural development.</a> Mol Cell Proteomics. 10 (5), (2011).</li><li>Ramirez, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+Analysis+of+the+Ubiquitin+Landscape+in+the+Drosophila+Embryonic+Nervous+System+and+the+Adult+Photoreceptor+Cells.">Proteomic Analysis of the Ubiquitin Landscape in the Drosophila Embryonic Nervous System and the Adult Photoreceptor Cells.</a> PLoS One. 10 (10), 0139083 (2015).</li></ol>

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 ...

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 (AP-MS) has proven to be a powerful approach to study protein-protein interactions (PPIs) in their native environment in cells and tissues1,2. This method allows for the identification of direct or indirect interactions at specific developmental stages or tissue contexts, and has been successfully used to identify multiple novel PPIs in a variety of developmental pathways (reviewed in reference1). Despite the undisputed success of PPI studies, most of them have been carried out in cultured cells, in which the &#34;bait&#34; proteins of interest were overexpressed. There are two issues with studying PPIs in cell culture: first, a specific cell line may not provide a full complement of interactions due to lack of expression of certain proteins. Second, high overexpression usually employed in such analyses might lead to artefacts such as protein misfolding or identification of false positive interactions.
Both of these limitations can be overcome by analyzing PPIs in vivo. A limiting step in such experiments is the availability of the starting material for purifying protein complexes. Drosophila melanogaster has long been used as a model for functional analysis, and recently it has also been shown to be an excellent system for studying PPIs in vivo. Drosophila embryogenesis represents a particularly attractive tissue type to study PPIs, because embryos can be easily collected in large quantities, and also because most genes (&#62;88%) are expressed over the course of embryogenesis, thus providing a rich in vivo environment for detecting relevant PPIs3.
Traditionally, biochemical studies in flies utilized very large-scale embryo collections (100-150 g), such as those necessary for purifying functional transcriptional lysates4,5. Previous AP-MS studies in Drosophila also needed large amounts of embryos (5-10 g), because they relied on a two-step purification approach such as tandem affinity purification (TAP), with the associated loss of material at each step6. Large amounts of starting material necessitated setting up embryo collections in large population cages, which can be both expensive (when purchased commercially) and time-consuming to maintain and clean7,8,9.
Recent advances in the development of single-step affinity purification approaches, as well as the increasing sensitivity of mass spectrometers, have reduced the necessary amount of starting material by an order of magnitude. Using tags such as the streptavidin-binding peptide (SBP) or green fluorescent protein (GFP) and starting from less than 1 g of embryos, it is possible to isolate the amounts of the bait protein and interacting components that would be sufficient for identification by mass spectrometry10,11.
The goal of the protocol presented here is to help the researchers overcome a perceived barrier to biochemical analysis of PPIs in vivo. To that end, we provide a simple and inexpensive procedure to collect Drosophila embryos at medium scale (0.5-1 g), followed by one-step preparation of whole-cell protein extracts that are suitable for subsequent analysis by AP-MS or other approaches. Our method relies on the use of custom-made 1-L or 5-L population cages that can be easily produced by any laboratory. Furthermore, the extraction conditions presented here have been validated in several studies, both in cultured cells and in vivo10,12,13,14,15,16,17.

<table border="0" cellpadding="0" cellspacing="0" width="1384">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Leaktite 5-Qt. Natural Multi Mix Container (Pack of 3)</td>
			<td style="width:183px;">Home Depot</td>
			<td style="width:119px;">209330</td>
			<td style="width:644px;">For making 5-L cages.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Leaktite 5-Qt. Natural Multi Mix Lid (Pack of 3)</td>
			<td style="width:183px;">Home Depot</td>
			<td style="width:119px;">209320</td>
			<td style="width:644px;">Diameter 8.37 in. Lids for 5-L cages.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Fisherbrand Petri dishes with clear lid, 100 x 15 mm</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:119px;">FB0875712</td>
			<td style="width:644px;">Material: Polystyrene. To be used with 1-L cages.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Fisherbrand Petri dishes with clear lid, 150 x 15 mm</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:119px;">FB0875714</td>
			<td style="width:644px;">Material: Polystyrene. To be used with 5-L cages.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Fisherbrand Petri dishes with clear lid, 60 x 15 mm</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:119px;">FB0875713A</td>
			<td style="width:644px;">Material: Polystyrene. Used during embryo dechorionation.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Sefar NITEX nylon mesh</td>
			<td style="width:183px;">Sefar</td>
			<td style="width:119px;">03-180/44</td>
			<td style="width:644px;">180 micron, 44% open area, used for fly cages.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Milwaukee 3 in. Hole Dozer Hole Saw with Arbor</td>
			<td style="width:183px;">Home Depot</td>
			<td style="width:119px;">49-56-9670</td>
			<td style="width:644px;">3 inch cutting tool for making 1-L cages.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:439px;">Nalgene Wide-Mouth Straight-Sided PMP Jars with White Polypropylene Screw Closure</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:119px;">11-823-33</td>
			<td style="width:644px;">For making 1-L cages. Thermo Scientific cat # 21171000. Alternative 1-L containers can be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Red Star Active Dry Yeast, 2 pound pouch</td>
			<td style="width:183px;">Red Star&#160;</td>
			<td style="width:119px;"></td>
			<td style="width:644px;">can be purchased from Amazon.com or other suppliers.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Frozen 100% Apple Juice Concentrate&#160;</td>
			<td style="width:183px;"></td>
			<td style="width:119px;"></td>
			<td style="width:644px;">can be purchased from a grocery store. Has to say &#34;100% apple juice&#34; on the can.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Methyl 4-hydroxybenzoate</td>
			<td style="width:183px;">Acros Organics</td>
			<td style="width:119px;">AC126965000</td>
			<td style="width:644px;">also known as methylparaben or Tegosept. Used as a preservative in AJ plates.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">IGEPAL CA-630 for molecular biology, 100 ml</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:119px;">I8896</td>
			<td style="width:644px;">used for preparing lysis buffer.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:439px;">Nalgene Rapid-Flow Sterile Disposable Filter Units with CN Membrane&#160;</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:119px;">09-740-2A</td>
			<td style="width:644px;">0.2 &#956;m pore size. Used for filtering 5x lysis buffer. Thermo Scientific cat # 1260020.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">CO-RO ROCHE cOmplete Protease Inhibitor Cocktail</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:119px;">11697498001</td>
			<td style="width:644px;">Vial of 20 tablets. Used for protease inhibition in lysis buffer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Corning SFCA syringe filters&#160;</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:119px;">09-754-21</td>
			<td style="width:644px;">SFCA membrane, diameter 26 mm, pore size 0.45 &#956;m. Used for filtering final extract samples.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">BD&#160;Luer-Lok Disposable Syringes without Needles</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:119px;">14-823-2A</td>
			<td style="width:644px;">for filtering final extract samples. BD cat # 309604.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:439px;">Bleach</td>
			<td style="width:183px;">Clorox</td>
			<td style="width:119px;"></td>
			<td style="width:644px;">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.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Corning Costar Netwell Plates</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:119px;">07-200-213</td>
			<td style="width:644px;">mesh containers for embryo collections. 74 &#956;m mesh size, 6-well.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Wheaton Dounce Tissue Grinders, capacity 15 ml</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:119px;">06-435B</td>
			<td style="width:644px;">homogenizer for embryos, with loose and tight pestles. Wheaton cat # 357544.</td>
		</tr>
	</tbody>
</table>

medium-scale preparation of drosophila embryo extracts for proteomic experiments

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.

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) promoter18,19. The arm promoter is active during most stages of Dr...

Watch this Scientific Journal Video about Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments at JoVE.com

Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments

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).

Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments

Analysis of protein-protein interactions (PPIs) has become an indispensable approach to study biological processes and mechanisms, such as cell signaling, ...

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