Multi-component protein complexes play crucial roles during cellular function and development. Here we describe a method used to isolate native protein complexes from Drosophila embryos after in vivo crosslinking followed by purification of the crosslinked complexes for subsequent structure-function analysis.
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Method Article
An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos
In This Article
Summary
Abstract
Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to assemble. Therefore, to identify these functional protein complexes, it is important to stabilize them in vivo before cell lysis and subsequent purification. Here we describe a method used to isolate large bona fide protein complexes from Drosophila embryos. This method is based on embryo permeabilization and stabilization of the complexes inside the embryos by in vivo crosslinking using a low concentration of formaldehyde, which can easily cross the cell membrane. Subsequently, the protein complex of interest is immunopurified followed by gel purification and analyzed by mass spectrometry. We illustrate this method using purification of a Tudor protein complex, which is essential for germline development. Tudor is a large protein, which contains multiple Tudor domains - small modules that interact with methylated arginines or lysines of target proteins. This method can be adapted for isolation of native protein complexes from different organisms and tissues.
Introduction
Isolation of multisubunit protein assemblies and DNA- or RNA-protein complexes is performed to identify protein complexes, genomic loci recognized by DNA-binding regulatory proteins or RNA targets of RNA binding proteins. Different methods allow genome-wide identification of DNA sites recognized by transcription factors or chromatin proteins (ChIP-seq)1 and RNA targets associated with a given RNA-binding protein (CLIP-seq)2. The libraries of RNA-derived cDNAs or DNA targets are then deeply sequenced. These methods use chemical or UV-induced cross-linking to stabilize the complexes followed by immunoprecipitation (IP) with an antibody against a pr....
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Protocol
1. Preparing Large Apple Juice-agar Plates
- To make 4 plates, add 375 ml H2O, 11.25 g fly agar and a stir bar to a 1,000 ml flask. This is Mix A. Autoclave Mix A with the flask lid loosely capped on one 30 min-sterilization cycle for liquid goods.
- Add 125 ml apple juice, 12.5 g table sugar and a stir bar to a 500 ml beaker. This is Mix B. Heat Mix B on a heated platform while stirring and maintain the temperature at approximately 70 °C until the autoclaving of Mix A is finished.
- Upon the completion of Mix A autoclaving, transfer Mix B to Mix A. Keep stirring the combined mixture gently and let it cool for 15 min....
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Results
The effectiveness of the crosslinking and the successful purification of the crosslinked Tud protein complex were analyzed by SDS-PAGE on a 3% - 7% step gel (illustrated in Figure 1) followed by western blot (Figure 2).
The purpose of using the 3% - 7% step gel is based on the effective separation of crosslinked Tud protein complex from the remaining uncrosslinked Tud protein and concentration of the complex. Under our in vivo crosslinking conditions .......
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Discussion
Formaldehyde has been commonly used as a crosslinking reagent for identifying protein-protein and protein-nucleic acid interactions. Its good solubility and cell membrane permeability, together with the compatibility with downstream mass spectrometry procedures, make formaldehyde an ideal candidate agent for intracellular crosslinking applications3,15-17. In particular, it was successfully used to identify mRNAs associated with Vasa, a critical germ cell RNA helicase in Drosophila11. In add.......
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Disclosures
The authors have nothing to disclose.
Acknowledgements
We thank Jordan Davis, Yanyan Lin, Eric Schadler and Jimiao Zheng for their technical help with this study. This work was supported by NSF CAREER grant MCB-1054962 to A.L.A.
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| Drosophila agar | Lab Scientific (http://www.labscientific.com/) | FLY-8020-1 | Do not autoclave the water and agar mix for prolonged period of time as it will cause the apple juice plates to become fragile |
| Methyl 4-hydroxybenzoate | Sigma (http://www.sigmaaldrich.com/united-states.html) | H5501 | Other name: TEGOSEPT |
| Population cage | Flystuff (http://www.flystuff.com/) | 59-104 | |
| Fine nylon mesh | Flystuff (http://www.flystuff.com/) | 57-102 | When making the collection basket, cut the mesh slightly larger than the opening of the falcon tube cap to ensure a tight seal |
| Dounce homogenizer | Sigma (http://www.sigmaaldrich.com/united-states.html) | D8938-1SET | Chill the homogenizer on ice and prerinse with cold lysis buffer before use to prevent protein degradation |
| Protease inhibitor cocktail | Roche (http://www.rocheusa.com/portal/usa) | 4693132001 | PBS could be used to prepare concentrated protease inhibitor stock solution |
| Anti-HA agarose beads | MBL international (http://www.mblintl.com/) | 561-8 | The kit also includes HA-peptide and spin columns |
| HA-peptide | MBL international (http://www.mblintl.com/) | 561-8 | Prepare to 2 mg/ml with PBS |
| Spin Column | MBL international (http://www.mblintl.com/) | 561-8 | Spin columns are included as part of the kit |
| Isopropanol | Fisher Scientific (www.fishersci.com/) | BP26324 | |
| Triton X-100 | Fisher Scientific (www.fishersci.com/) | BP151-500 | |
| Heptane | Fisher Scientific (www.fishersci.com/) | H350-4 | |
| PBS | Invitrogen (https://www.lifetechnologies.com/us/en/home.html) | AM9625 | Dilute from 10X to 1X with nanopure water before use |
| Formaldehyde | Fisher Scientific (www.fishersci.com/) | BP531-500 | |
| Glycine | BioRad (www.bio-rad.com/) | 161-0724 | |
| SDS | BioRad (www.bio-rad.com/) | 161-0301 | |
| Urea | BioRad (www.bio-rad.com/) | 161-0730 | |
| Phenylmethanesulfonyl fluoride | Sigma (http://www.sigmaaldrich.com/united-states.html) | P7626-1G | Prepare 200 mM stock solution in isopropanol then dilute to working concentration of 2 mM in lysis buffer |
| IGEPAL CA-630 | Sigma (http://www.sigmaaldrich.com/united-states.html) | I8896-50ML | |
| Tween 20 | Fisher Scientific (www.fishersci.com/) | BP337-100 | |
| 15-ml tubes | USA Scientific (http://www.usascientific.com/) | 1475-0511 | |
| Bleach | Clorox brand | Dilute with equal volume of nanopure water to make 50% bleach | |
| 50-ml tubes | BD Biosciences (http://www.bdbiosciences.com/home.jsp) | 352098 | |
| Top layer sieve | Fisher Scientific (www.fishersci.com/) | 04-884-1AK | |
| Bottom layer sieve | Fisher Scientific (www.fishersci.com/) | 04-884-1BA |
References
- Landt, S. G., et al. ChIP-seq guidelines and practices of the ENCODE and modENCODE consortia. Genome research. 22, 1813-1831 (2012).
- Murigneux, V., Sauliere, J., Roest Crollius, H., Le Hir, H. Transcriptome-wide identification of RNA binding sites by CLIP-seq. Meth....
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