This work was in part supported by Kansas INBRE, P20GM103418 to Li and Zinovyeva and R35GM124828 to Zinovyeva. We thank Min Han for generously sharing the anti-AIN-1 antibody. Some of the strains used in the course of this work were provided by the Caenorhabditis Genetics Center (CGC), funded by NIH Office of Research Infrastructure Programs (P40 OD010440).

1. Worm sample collection
<ol>
	<li>Seed mixed stage or synchronized13 worms on NGM solid plates at the required temperature and allow the worms to grow until the desired stage. For basic C. elegans growth and maintenance, please see Stiernagle et al. and Porta-de-la-Riva et al.14,13.</li>
	<li>Collect worms in a 15 mL conical centrifuge tube by washing the worm plates with M9 buffer.</li>
	<li>Pellet the worms by centrifuging at 400 x g at room temperature (RT) for 2 min and discard the supernatant. 
	NOTE: The worm pellet size for extract preparation is between 100 &#181;L and 500 &#181;L. A 300 &#181;L pellet of packed worms is recommended for downstream immunoprecipitation experiments and typically yields ~4.5 mg of total protein, while a 500 &#181;L pellet will yield ~7.5 mg of total protein.</li>
	<li>Perform additional 3&#8211;5 washes with M9 buffer (see Table 1) or until the supernatant is no longer cloudy.</li>
	<li>Perform one final wash with ddH2O.</li>
	<li>Move the loose worm pellet to a 1.5 mL microcentrifuge tube and spin down at 400 x g at RT for 2 min. Discard the remaining supernatant to obtain a packed worm pellet and proceed to extract preparation. 
	NOTE: The protocol can be paused here. Worm pellets may be flash frozen in liquid nitrogen immediately and stored at -80 &#176;C or in liquid nitrogen. Please note that worm pellets can only be thawed once and cannot be refrozen.</li>
</ol>
2. Extract preparation of the worm pellet
NOTE: The extract preparation should be performed on ice or at 4 &#176;C.
<ol>
	<li>If frozen, thaw worm pellet on ice. 
	NOTE: If the desired packed worm pellet size of 300 &#181;L was not obtained&#160;during sample collection, multiple smaller pellets can be combined until enough material is present for further extraction.</li>
	<li>Add an equal volume of ice-cold 2x lysis buffer (60 mM HEPES, pH = 7.4, 100 mM potassium chloride, 0.1% Triton X, 4 mM magnesium chloride, 10% glycerol, 2 mM DTT with RNase inhibitor, protease inhibitor, and phosphatase inhibitors; see Table 1) and vortex or pipette up and down to mix. Spin the tube(s) down to collect the mixture at the bottom of the tube.</li>
	<li>Move the mixture into a 1.5 mL RNase-free tube containing metal beads (see Table of Materials) and put the sample in the bead mill homogenizer (see Table of Materials) at 4 &#176;C. Ensure that the tube caps are tightened and the samples are balanced inside the homogenizer.</li>
	<li>Homogenize the sample at the highest speed (setting 12) for 4 min.</li>
	<li>Remove the sample from the beads and place it into a new 1.5 mL microcentrifuge tube. Alternatively, a magnet provided with the homogenizer can be used to remove the beads from the sample.</li>
	<li>Spin down the extract at 19,000 x g for 20 min at 4 &#176;C to clarify the protein extract.</li>
	<li>Transfer the supernatant into a fresh 1.5 mL tube on ice, while avoiding carry-over of the white, cloudy precipitate that forms on top of the sample. The supernatant is now the clarified extract. 
	NOTE: Save 10 &#181;L of the clarified extract to determine the total protein concentration.</li>
	<li>Use the extract immediately for the following experiments or flash freeze the extract in liquid nitrogen and store at -80 &#176;C. 
	NOTE: The protocol may be paused here. Extracts may be stored at ultralow temperature (-80 &#176;C freezer or liquid nitrogen for ~6 months). Frozen extracts may be thawed once and cannot be refrozen.</li>
	<li>Determine the total protein concentration of the extract using a protein concentration assay kit compatible with detergents (see Table of Materials) according to the manufacturer&#8217;s instructions.</li>
</ol>
3. Immunoprecipitation
NOTE: All the immunoprecipitation steps for extract preparation should be performed on ice or at 4 &#176;C. It is recommended to use 2 mg of total protein for each immunoprecipitation. However, successful immunoprecipitations with 0.8&#8211;1 mg of total protein have been performed. Always use fresh or freshly thawed protein extracts. The following protocol is outlined to perform immunoprecipitation from 2 mg of total protein, or a single immunoprecipitation experiment. The amount of beads and antibody may be increased or decreased accordingly for multiple samples or if a different amount of protein extract is used.
<ol>
	<li>Place or thaw the protein extract on ice. The sample can be diluted to 10 mg/mL or 5 mg/mL with ice-cold 1x lysis buffer (see Table 1).</li>
	<li>Resuspend the magnetic beads by inversion and transfer 150 &#181;L of the 50% beads suspension into a 1.5 mL tube. Magnetize the beads on ice against a magnetic stand for 1 min or until the solution is clear. Discard the supernatant.</li>
	<li>Remove the tube from the magnetic stand and wash the beads in 1x lysis buffer using 2 volumes (i.e., 300 &#181;L) of bead slurry. Repeat the wash 2x for a total of three washes.</li>
	<li>Resuspend the beads in 150 &#181;L of ice-cold lysis buffer.</li>
	<li>Transfer 75 &#181;L of the bead slurry to 2 mg of protein extract and incubate at 4 &#176;C for 1 h with gentle agitation. Save the remaining bead suspension on ice for later use. 
	NOTE: This step is performed to reduce nonspecific protein binding to beads during the immunoprecipitation step.</li>
	<li>Place the tube with sample on the magnetic stand on ice for 1 min or until the beads are fully magnetized and the sample is clear. Transfer the supernatant to a new 1.5 mL tube; do not disturb the beads. This is the precleared protein lysate. Save 10% of the sample for Western blot analysis.</li>
	<li>Add 20 &#181;g of affinity purified antibody to the precleared lysate and incubate at 4 &#176;C for 1 h with gentle agitation. 
	NOTE: The amount of antibody used for immunoprecipitation is specific to the antibody and protein and should be empirically determined to ensure effective immunoprecipitation of the target protein.</li>
	<li>Add the remaining 75 &#181;L of the prewashed beads suspension (step 3.5) to the antibody/lysate mixture and incubate for 1 h at 4 &#176;C with gentle agitation.</li>
	<li>Place the tube in the magnetic stand on ice for 1 min or until the beads are fully magnetized and the sample is clear. Save the supernatant for Western blot analysis (optional).</li>
	<li>Wash the beads containing the immunoprecipitate 3x in 450 &#181;L of wash buffer (30 mM HEPES, pH = 7.4, 100 mM potassium chloride, 0.1% Triton X, 2 mM magnesium chloride, 10% glycerol, 1 mM DTT; see Table 1) on ice. 
	NOTE: Additional washes may be performed if more stringent washing conditions are preferred.</li>
	<li>Resuspend the bead pellet in 20 &#181;L of 2x SDS/BME protein gel loading buffer (see Table of Materials) and denature by boiling at 95 &#176;C for 5 min prior to loading onto an SDS-PAGE gel. Alternatively, denatured samples can be stored at -20 &#176;C for several months. 
	NOTE: A portion of the bead immunoprecipitate can be saved for downstream RNA isolation, if desired.</li>
</ol>
4. Western blot detection of IP samples
<ol>
	<li>Load the IP samples onto the SDS-PAGE gel (see Table of Materials). Avoid transferring the beads by placing the IP tubes on the magnetic stand for 1 min prior to aspiration of the sample.</li>
	<li>Perform the Western blotting15,16 and antibody staining16 with the following modifications: dilute the ALG-1 antibody17 1:500 in 5% non-fat dry milk (NFDM); dilute the HRPK-1 antibody2 1:1,000 in 5% NFDM; and dilute the AIN-1 antibody18 1:10,000 in 5% NFDM. Secondary antibodies (see Table of Materials) were used according to the manufacturer&#8217;s instructions.</li>
	<li>Detect the bands with HRP-based chemiluminescence (see Table of Materials).</li>
</ol>

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The authors have nothing to disclose.

C. elegans is an excellent model for studying fundamental questions in cell, molecular, and developmental biology19. In addition to its power as a genetic model system, C. elegans is amenable to biochemical approaches, including, but not limited to, protein immunoprecipitation and co-immunoprecipitation. One potential hurdle when conducting immunoprecipitation experiments is lack of antibodies specific to the proteins of interest. If no antibody is available, custom polyclonal or monoclonal antibodies can be generated. However, recent innovations in genome editing technology have allowed researchers to rapidly introduce mutations or tag endogenous C. elegans genes20,21, facilitating studies that unravel the genetic, functional, and physical interactions among the genes and the encoded proteins. Specifically, CRISPR/Cas9-mediated tagging of C. elegans genes at the endogenous loci has reduced the dependence of immunoprecipitation experiments on antibody availability, making co-immunoprecipitation experiments much more feasible. C. elegans genes can be tagged with a variety of tags ranging from fluorescent tags such as GFP or mCherry to small tags such as FLAG and HA. Antibodies recognizing these tags are readily available commercially, facilitating the studies of protein-protein interactions via immunoprecipitation approaches.
The presented protocol, outlined in Figure 1, can be performed for a small number of samples or scaled up, allowing for up to 24 sample preparations at a time. While the initial characterizations of protein-protein interactions via immunoprecipitation are typically done in wild type backgrounds under normal growing conditions, follow-up studies frequently necessitate testing the protein-protein interactions in a variety of genetic backgrounds or under different growth conditions. The ability to simultaneously prepare multiple extracts saves time and, importantly, ensures extract preparation consistency among the different samples. A negative control is always required, with the ideal control being a null mutation in the gene encoding for the immunoprecipitated protein of interest (See Figure 3 and Figure 4 for examples).
This extract protocol allows for rapid protein extract preparation from C. elegans samples and is comparable to zirconium bead-based homogenization8. Bead homogenization in general can be scaled up to multiple simultaneous sample preparations using a variety of bead mill homogenizers or similar equipment. Some more economical bead mill homogenizers may reduce the number of samples that can be processed simultaneously, however. Alternatively, the presented extract protocol is compatible with dounce-based extract preparation, which represents an economical alternative. While different bead mill homogenizers were not tested, most&#160;are likely to be compatible with this protein extract protocol, as long as complete disruption of the C. elegans samples is achieved.
As presented, this extract preparation protocol is compatible with multiple downstream experiments, including protein immunoprecipitation2 and microRNA pull-down12 and allows for downstream collection of both protein and RNA components. It also efficiently extracts both nuclear and cytoplasmic proteins (Figure 2, Figure 3, and Figure 4). Similarly, the presented immunoprecipitation protocol permits RNA isolation from protein-associated immunoprecipitates. While the immunoprecipitation protocol was originally developed to identify ALG-1 protein interactors, the method can be adapted to test for interactions between any proteins of interest. In fact, the immunoprecipitation conditions used worked equally well for immunoprecipitating ALG-1 (Figure 3) and HRPK-1 (Figure 4). This protocol is an excellent starting point for immunopurification of RNA-binding proteins. It should be noted, however, that some changes in buffer composition may be required for other proteins of interest. The changes may depend on the physical and biochemical properties of the protein of interest and must be implemented on a case-by-case basis.
Once the target protein (here, ALG-1 or HRPK-1) is immunoprecipitated, Western blotting can be used to test the co-immunoprecipitate for specific protein interactors.
Alternatively, the copurified immunoprecipitate can be subjected to mass spectrometry analysis to identify all the putative interacting proteins. Confirmed co-immunoprecipitation interactions can then be examined in a variety of genetic backgrounds or conditions to identify potential regulation of the specific interaction. For example, to determine whether hrpk-1 plays a role in ALG-1/AIN-1 miRISC assembly, ALG-1-AIN-1 coprecipitation was assessed both in a wild type background and in the absence of HRPK-1 (Figure 3). hrpk-1 was found to be dispensable for ALG-1/AIN-1 interaction2 (Figure 3). In addition, CRISPR/Cas9 genome editing technology can be employed to generate single point or domain deletion mutations in the proteins of interest. Retesting the ability of the generated mutants to coprecipitate with their protein interactors can reveal which domains or residues mediate the physical interaction. Such future studies can yield invaluable information about the mechanism of protein function and regulation. These approaches, combined with the power of C. elegans genetics, can provide important insights into the fundamental molecular processes that govern animal development and cellular function.

Identifying the macromolecular interactions of a protein of interest can be key to learning more about its function. Immunoprecipitation and co-immunoprecipitation experiments can be used to identify the entire interactome of a protein through large-scale proteomic approaches1 or to specifically test a protein&#8217;s ability to coprecipitate with a hypothesized interactor. In C. elegans, both methods have been successfully employed to learn more about the activity of a variety of proteins, including those that closely function with microRNAs to regulate gene expression2,3,4. Co-immunoprecipitation experiments have the advantage of testing the protein-protein interactions in their native cellular environment, but extract preparation can be challenging and time-consuming. Efficient lysis of the sample is necessary, but care must be taken to minimize the disruption of protein-protein interactions. Methods such as douncing5, sonication6, Balch homogenization7, and zirconia beads-homogenization8,9 have been used to successfully prepare C. elegans total protein extracts. These methods, with the exception of&#160;zirconia bead homogenization, have limitations in terms of the number of samples that can be processed simultaneously. Presented is an alternative method that can be easily scaled up to allow for high-throughput, rapid protein extract preparation from C. elegans samples followed by co-immunoprecipitation. Specifically, the method can prepare up to 24 samples at a time, greatly reducing the time required for extract preparation. By contrast, for example, douncing typically allows for only one sample preparation at a time. This extract method can be used to prepare extracts from any developmental stage of C. elegans.
Described is a step-by-step procedure for animal sample collection, extract preparation, immunoprecipitation, and presentation of Western blotting data to confirm successful protein pulldown and detection of the co-immunoprecipitating protein of interest. To demonstrate the effectiveness of the protocol, two co-immunoprecipitation experiments were performed between&#160;1) microRNA Argonaute ALG-1 and AIN-1, a GW182 homolog; and 2) ALG-1 and HRPK-1, a newly identified ALG-1 interactor2. ALG-1 and AIN-1 are core proteins that comprise the microRNA-induced silencing complex (miRISC) and&#160;the interaction between these two proteins is well established10,11. The extract preparation protocol was effective in the ALG-1-AIN-1 co-immunoprecipitation experiment. This protocol also successfully confirmed the interaction between ALG-1 and its newly identified interactor, HRPK-12.
In summary, the manuscript describes a C. elegans extract preparation protocol that can be&#160;scaled up to simultaneously process 24 samples along with a co-immunoprecipitation protocol that can be used to&#160;identify new or confirm hypothesized interactions between proteins. The extract preparation protocol is compatible with a number of downstream experiments, including protein immunoprecipitation2&#160;and&#160;microRNA pulldowns12. Furthermore, the immunoprecipitation protocol can be adapted to test for interactions between any two or more endogenous, endogenously tagged, or overexpressed C. elegans proteins in a variety of genetic backgrounds.

<table><tbody><tr><td>15 mL tube</td><td>VWR</td><td>89039-664</td><td>STEP 1.2</td></tr><tr><td>2x Laemmli Sample Buffer</td><td>BioRed</td><td>1610737</td><td>STEP 3.11</td></tr><tr><td>4&amp;ndash;20% Mini-PROTEAN TGX Precast Protein Gels</td><td>BioRed</td><td>4561096</td><td>STEP 4.1</td></tr><tr><td>anti-AIN-1 monoclonal antibody</td><td>custom generated</td><td>n/a</td><td>STEP 4.2, see ref. Zhang et al. 2007</td></tr><tr><td>anti-ALG-1 monoclonal antibody</td><td>custom generated by PRF&amp;amp;L</td><td>n/a</td><td>STEP 4.2</td></tr><tr><td>anti-HRPK-1 monoclonal antibody</td><td>custom generated by PRF&amp;amp;L</td><td>n/a</td><td>STEP 4.2</td></tr><tr><td>Bullet Blender Storm Homogenizer</td><td>MidSci</td><td>BBY24M</td><td>STEP 2.3</td></tr><tr><td>DL-Dithiothreitol (DTT)</td><td>Sigma</td><td>D9779-5G</td><td>Table 1</td></tr><tr><td>Dynabeads Protein A for Immunoprecipitation</td><td>Thermo Fisher</td><td>10002D</td><td>STEP 3.2</td></tr><tr><td>DynaMag-2 Magnet</td><td>Thermo Fisher</td><td>12321D</td><td>STEP 3.2</td></tr><tr><td>EDTA-free protease inhibitors</td><td>Roche</td><td>11836170001</td><td>Table 1</td></tr><tr><td>GFP antibody (FL)</td><td>Santa Cruz Biotechnology</td><td>sc-8334</td><td>Figure 2</td></tr><tr><td>Glycerol</td><td>Thermo Fisher</td><td>G33-500</td><td>Table 1</td></tr><tr><td>Goat Anti-Rabbit Secondary Antibody, HRP</td><td>BioRed</td><td>1662408</td><td>STEP 4.2</td></tr><tr><td>Goat anti-Rat IgG (H+L) Secondary Antibody, HRP</td><td>Thermo Fisher</td><td>31470</td><td>STEP 4.2</td></tr><tr><td>HEPES</td><td>Sigma</td><td>H4034-500G</td><td>Table 1</td></tr><tr><td>LICOR WesternSure PREMIUM Chemiluminescent Substrate, 100 mL Kit</td><td>LI-COR</td><td>926-95000</td><td>STEP 4.3</td></tr><tr><td>Magnesium chloride hexahydrate ACS</td><td>VWR</td><td>VWRV0288-500G</td><td>Table 1</td></tr><tr><td>Magnesium Sulfate Anhydrous</td><td>Thermo Fisher</td><td>M65-500</td><td>Table 1</td></tr><tr><td>Microcentrifuge Tubes, 1.5 mL</td><td>VWR</td><td>20170-333</td><td>STEP 1.6</td></tr><tr><td>N2 wild type</td><td>CGC</td><td></td><td></td></tr><tr><td>Navy RINO RNA Lysis Kit 50 pack (1.5 mL)</td><td>MidSci</td><td>NAVYR1-RNA</td><td>STEP 2.3</td></tr><tr><td>Phosphatase inhibitor cocktail 2</td><td>Sigma</td><td>P5726-1ML</td><td>Table 1</td></tr><tr><td>Phosphatase inhibitor cocktail 3</td><td>Sigma</td><td>P0044-1ML</td><td>Table 1</td></tr><tr><td>Potassium Chloride</td><td>Thermo Fisher</td><td>P217-500</td><td>Table 1</td></tr><tr><td>Potassium phosphate monobasic</td><td>Thermo Fisher</td><td>P285-3</td><td>Table 1</td></tr><tr><td>RC DC Protein Assay Kit I</td><td>BioRed</td><td>5000121</td><td>STEP 2.9</td></tr><tr><td>RNaseOUT Recombinant Ribonuclease Inhibitor</td><td>Thermo Fisher</td><td>10777019</td><td>Table 1</td></tr><tr><td>Sodium Chloride</td><td>Thermo Fisher</td><td>S271-500</td><td>Table 1</td></tr><tr><td>Sodium Phosphate Dibasic Anhydrous</td><td>Thermo Fisher</td><td>S374-500</td><td>Table 1</td></tr><tr><td>TritionX-100</td><td>Sigma</td><td>X100-500ML</td><td>Table 1</td></tr><tr><td>UY38 hrpk-1(zen17)</td><td>available upon request</td><td></td><td></td></tr><tr><td>VT1367 col-19::gfp(maIS105)</td><td>available upon request</td><td></td><td></td></tr><tr><td>VT3841 alg-1(tm492)</td><td>available upon request</td><td></td><td></td></tr></tbody></table>

protein extract preparation and co-immunoprecipitation from caenorhabditis elegans

Co-immunoprecipitation methods are frequently used to study protein-protein interactions. Confirmation of hypothesized protein-protein interactions or identification of new ones can provide invaluable information about the function of a protein of interest. Some of the traditional methods for extract preparation frequently require labor-intensive and time-consuming techniques. Here, a modified extract preparation protocol using a bead mill homogenizer and metal beads is described as a rapid alternative to traditional protein preparation methods. This extract preparation method is compatible with downstream co-immunoprecipitation studies. As an example, the method was used to successfully co-immunoprecipitate&#160;C. elegans microRNA Argonaute ALG-1 and two known ALG-1 interactors: AIN-1, and HRPK-1. This protocol includes descriptions of animal sample collection, extract preparation, extract clarification, and protein immunoprecipitation. The described protocol can be adapted to test for interactions between any two or more endogenous, endogenously tagged, or overexpressed C. elegans proteins in a variety of genetic backgrounds.

This protocol (schematized in Figure 1) was used successfully to obtain C. elegans total protein extracts (Figure 2)&#160;for downstream immunoprecipitation of several proteins2 (Figure 3 and Figure 4). The presented bead mill homogenizer protocol was comparable in total protein extraction to dounce-based methods (Figure 2) and efficiently extracted nuclear (COL-19::GFP(NLS) (Figure 2) and cytoplasmic proteins (Figure 3 and Figure 4). Multiple samples of various sizes were extracted simultaneously (Figure 2). Argonaute proteins interact&#160;with members of the GW182 protein family, forming the miRISCs that bind to the target messenger RNAs and repress their expression10. Figure 3 shows successful co-immunoprecipitation of core miRISC components ALG-1 and AIN-1, consistent with previous reports11,17. More recently, efforts were made to identify additional protein interactors of Argonaute ALG-13 in order to learn more about how microRNA biogenesis and activity might be regulated by auxiliary factors. The RNA-binding protein HRPK-1 was identified in ALG-1 immunoprecipitates3. This interaction was recently confirmed in a reciprocal HRPK-1 immunoprecipitation experiment2. The presented extract and immunoprecipitation protocols&#160;successfully recovered ALG-1 in HRPK-1-specific co-immunoprecipitates (Figure 4). In addition, the ALG-1&#8212;AIN-1 interaction was tested in a variety of genetic backgrounds and HRPK-1 was shown to be unnecessary for the ALG-1/AIN-1 miRISC assembly2 (Figure 3). Supplemental figures are provided to show the full membrane probed (Supplemental Figure 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61243/61243fig1.jpg" /> 
Figure 1: Workflow schematic for C. elegans extract preparation and immunoprecipitation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61243/61243fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61243/61243fig2.jpg" /> 
Figure 2: Western blot comparison of a nuclear localized GFP, COL-19::GFP(NLS), levels in dounce-prepared and homogenized samples from 250 &#181;L and 100 &#181;L worm pellets. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61243/61243fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61243/61243fig3.jpg" /> 
Figure 3: GW182 homolog AIN-1 co-immunoprecipitates with ALG-1. Western blotting for ALG-1 and AIN-1 proteins in ALG-1 immunoprecipitates. The ALG-1/AIN-1 co-immunoprecipitation was not affected by the absence of hrpk-1. Input = 10% of IP. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61243/61243fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61243/61243fig4.jpg" /> 
Figure 4: ALG-1 co-immunoprecipitates with HRPK-1. Western blotting for HRPK-1 and ALG-1 in HRPK-1 immunoprecipitates is shown. Input = 10% of IP. * indicates antibody heavy chain. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61243/61243fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td colspan="2">M9 buffer (1 L)</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>3 g</td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>6 g</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>5 g</td>
		</tr>
		<tr>
			<td>1 M MgSO4</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>ddH2O</td>
			<td>up to 1 L</td>
		</tr>
		<tr>
			<td colspan="2">2x Lysis buffer (5 mL)</td>
		</tr>
		<tr>
			<td>HEPES (pH 7.4)</td>
			<td>200 &#181;L</td>
		</tr>
		<tr>
			<td>2 M KCl</td>
			<td>250 &#181;L</td>
		</tr>
		<tr>
			<td>10% TritonX</td>
			<td>100 &#181;L</td>
		</tr>
		<tr>
			<td>1 M MgCl2</td>
			<td>20 &#181;L</td>
		</tr>
		<tr>
			<td>100% glycerol</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>ddH2O</td>
			<td>up to 5 mL</td>
		</tr>
		<tr>
			<td>Add fresh:</td>
			<td></td>
		</tr>
		<tr>
			<td>1 M DTT</td>
			<td>20 &#181;L</td>
		</tr>
		<tr>
			<td>EDTA-free protease inhibitor</td>
			<td>1 tablet</td>
		</tr>
		<tr>
			<td>phosphatase inhibitor cocktail 2</td>
			<td>100 &#181;L</td>
		</tr>
		<tr>
			<td>phosphatase inhibitor cocktail 3</td>
			<td>100 &#181;L</td>
		</tr>
		<tr>
			<td colspan="2">1x Lysis buffer</td>
		</tr>
		<tr>
			<td colspan="2">Dilute 2x Lysis buffer with an equal volume of ddH20.</td>
		</tr>
		<tr>
			<td colspan="2">1x Wash buffer 10 mL)</td>
		</tr>
		<tr>
			<td>HEPES (pH 7.4)</td>
			<td>300 &#181;L</td>
		</tr>
		<tr>
			<td>2 M KCl</td>
			<td>500 &#181;L</td>
		</tr>
		<tr>
			<td>10% TritonX</td>
			<td>100 &#181;L</td>
		</tr>
		<tr>
			<td>1 M MgCl2</td>
			<td>20 &#181;L</td>
		</tr>
		<tr>
			<td>100% glycerol</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>ddH2O</td>
			<td>up to 10 mL</td>
		</tr>
		<tr>
			<td>1 M DTT</td>
			<td>20 &#181;L (add fresh)</td>
		</tr>
	</tbody>
</table>
Table 1: Recipes
Supplemental Figure 1. Full probed Western blot membranes used to generate Figures 2-4 are shown. (A) Probed membrane for Figure 2. Note that membrane was cut to allow for simultaneous probing for GFP and Tubulin, reducing the overall blot size. (B) Probed membrane for Figure 3. (C) Probed membrane for Figure 3. *denotes antibody heavy chain. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61243/Supplemental_Figure_1.pdf" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Protein Extract Preparation and Co-immunoprecipitation from Caenorhabditis elegans at JoVE.com

Protein Extract Preparation and Co-immunoprecipitation from Caenorhabditis elegans

This method describes a protocol for high-throughput protein extract preparation from Caenorhabditis elegans samples and subsequent co-immunoprecipitation.

Protein Extract Preparation and Co-immunoprecipitation from Caenorhabditis elegans

Co-immunoprecipitation methods are frequently used to study protein-protein interactions. Confirmation of hypothesized protein-protein interactions or ...

protein-extract-preparation-co-immunoprecipitation-from

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

8.7K Views.  Kansas State University. This method describes a protocol for high-throughput protein extract preparation from Caenorhabditis elegans samples and subsequent co-immunoprecipitation.

Video: Protein Extract Preparation and Co-immunoprecipitation from Caenorhabditis elegans

Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans

The changes in the plasma membrane localization of the epidermal growth factor receptor (EGFR) and its downstream effector RAS have been implicated in several diseases including cancer. The free-living nematode C. elegans possesses an evolutionary and functionally conserved EGFR-RAS-ERK MAP signal cascade which is central for the development of the vulva. Gain of function mutations in RAS homolog LET-60 and EGFR homolog LET-23 induce the generation of visible nonfunctional ectopic pseudovulva along the ventral body wall of these worms. Previously, the multivulval (Muv) phenotype in these worms has been shown to be inhibited by small chemical molecules. Here we describe a protocol for using the worm in a liquid-based assay to identify inhibitors that abolish the activities of EGFR and RAS proteins. Using this assay, we show R-fendiline, an indirect inhibitor of K-RAS, suppresses the Muv phenotype expressed in the let-60(n1046) and let-23(sa62) mutant worms. The assay is simple, inexpensive, is not time consuming to setup, and can be used as an initial platform for the discovery of anticancer therapeutics.

Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans

The genetically tractable nematode Caenorhabditis elegans can be used as a simple and inexpensive model for drug discovery. Described here is a protocol to identify anticancer therapeutics that inhibit the downstream signaling of RAS and EGFR proteins.

The changes in the plasma membrane localization of the epidermal growth factor receptor (EGFR) and its downstream effector RAS have been implicated in ...

Cancer Research

Isolation of Active Caenorhabditis elegans Nuclear Extract and Reconstitution for In Vitro Transcription

Caenorhabditis elegans has been an important model system for biological research since it was introduced in 1963. However, C. elegans has not been fully utilized in the biochemical study of biological reactions using its nuclear extracts such as in vitro transcription and DNA replication. A significant hurdle for using C. elegans in biochemical studies is disrupting the nematode&#39;s thick outer cuticle without sacrificing the activity of the nuclear extract. While several methods are used to break the cuticle, such as Dounce homogenization or sonication, they often lead to protein instability. There are no established protocols for isolating active nuclear proteins from larva or adult C. elegans for in vitro reactions. Here, the protocol describes in detail the homogenization of larval stage 4 C. elegans using a Balch homogenizer. The Balch homogenizer uses pressure to slowly force the animals through a narrow gap breaking the cuticle in the process. The uniform design and precise machining of the Balch homogenizer allow for consistent grinding of animals between experiments. Fractionating the homogenate obtained from the Balch homogenizer yields functionally active nuclear extract that can be used in an in vitro method for assaying transcription activity of C. elegans.

Isolation of Active Caenorhabditis elegans Nuclear Extract and Reconstitution for In Vitro Transcription

Here, we describe a detailed protocol for isolating active nuclear extract from larval stage 4 C. elegans and visualizing transcription activity in an in vitro system.

Caenorhabditis elegans has been an important model system for biological research since it was introduced in 1963. However, C. elegans has not been fully ...

Biochemistry

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, which are lipid signals derived from polyunsaturated fatty acids (PUFAs)10-14. These signalling molecules are difficult to study because of their low abundance and reactive nature. The characteristic feature of prostaglandins is a cyclopentane ring structure located within the fatty acid backbone. In mammals, prostaglandins can be formed through cyclooxygenase enzyme-dependent and -independent pathways10,15. C. elegans synthesizes a wide array of prostaglandins independent of cyclooxygenases6,16,17. A large class of F-series prostaglandins has been identified, but the study of eicosanoids is at an early stage with ample room for new discoveries. Here we describe a procedure for extracting and analyzing prostaglandins and other eicosanoids. Charged lipids are extracted from mass worm cultures using a liquid-liquid extraction technique and analyzed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The inclusion of deuterated analogs of prostaglandins, such as PGF2 &alpha;-d4 as an internal standard is recommended for quantitative analysis. Multiple reaction monitoring or MRM can be used to quantify and compare specific prostaglandin types between wild-type and mutant animals. Collision-induced decomposition or MS/MS can be used to obtain information on important structural features. Liquid chromatography mass spectrometry (LC-MS) survey scans of a selected mass range, such as m/z 315-360 can be used to evaluate global changes in prostaglandin levels. We provide examples of all three analyses. These methods will provide researchers with a toolset for discovering novel eicosanoids and delineating their metabolic pathways.

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

In this paper, we describe an optimized procedure for extracting and analyzing prostaglandins and other eicosanoids from C. elegans using LC-MS/MS.

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, ...

Biology

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These age-associated disorders are characterized by the appearance of protein aggregates with fibrillary structure in the brains of these patients. Exactly why normally soluble proteins undergo an aggregation process remains poorly understood. The discovery that protein aggregation is not limited to disease processes and instead part of the normal aging process enables the study of the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. Here we describe methodologies to examine inherent protein aggregation in Caenorhabditis elegans through complementary approaches. First, we examine how to grow large numbers of age-synchronized C. elegans to obtain aged animals and we present the biochemical procedures to isolate highly-insoluble-large aggregates. In combination with a targeted genetic knockdown, it is possible to dissect the role of a gene of interest in promoting or preventing age-dependent protein aggregation by using either a comprehensive analysis with quantitative mass spectrometry or a candidate-based analysis with antibodies. These findings are then confirmed by in vivo analysis with transgenic animals expressing fluorescent-tagged aggregation-prone proteins. These methods should help clarify why certain proteins are prone to aggregate with age and ultimately how to keep these proteins fully functional.

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

The goal of the method presented here is to explore protein aggregation during normal aging in the model organism C. elegans. The protocol represents a powerful tool to study the highly insoluble large aggregates that form with age and to determine how changes in proteostasis impact protein aggregation.

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture a full picture of the multiple levels of molecular processing and changes between different conditions. This protocol presents the method of isolating DNA, RNA, and protein from the same sample of the nematode Caenorhabditis elegans to remove the variation introduced when these biomolecules are isolated from replicates of similarly treated but ultimately different samples. Nucleic acids and protein are extracted from the nematode using the acid guanidinium thiocyanate-phenol-chloroform extraction method, with subsequent precipitation, washing, and solubilization of each. We show the successful isolation of RNA, DNA, and protein from a single sample from three strains of nematode and HeLa cells, with better protein isolation results in adult animals. Additionally, guanidinium thiocyanate-phenol-chloroform-extracted protein from nematodes improves the resolution of larger proteins, with enhanced detectable levels as observed by immunoblotting, compared to the traditional RIPA extraction of protein.
The method presented here is useful when investigating samples using a multiomic approach, specifically for the exploration of the proteome and transcriptome. Techniques that simultaneously assess multiomics are appealing because molecular signaling underlying complex biological phenomena is thought to occur at complementary levels; however, it has become increasingly common to see that changes in mRNA levels do not always reflect the same change in protein levels and that the time of collection is relevant in the context of circadian regulations. This method removes any intersample variation when assaying different contents within the same sample (intrasample.)

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

Here, we present a protocol for the isolation of RNA, DNA, and protein from the same sample, in an effort to reduce variation, improve reproducibility, and facilitate interpretations.

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture ...

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular vesicles (EVs) function outside the cell to regulate global physiological processes by transferring proteins, nucleic acids, metabolites, and lipids between tissues. EVs reflect the physiological state of their cells of origin. EVs are implicated to have fundamental roles in virtually every aspect of human health. Thus, EV protein and genetic cargos are being increasingly analyzed for biomarkers of health and disease. However, the EV field still lacks a tractable invertebrate model system that permits the study of EV cargo composition. C. elegans is well suited for EV research because it actively secretes EVs outside of its body into its external environment, permitting facile isolation. This article provides all the necessary information for generating, purifying, and quantifying these environmentally secreted C. elegans EVs including how to work quantitatively with very large populations of age-synchronized worms, purifying EVs, and a flow cytometry protocol that directly measures the number of intact EVs in the purified sample. Thus, the large library of genetic reagents available for C. elegans research can be tapped into for investigating the impacts of genetic pathways and physiological processes on EV cargo composition.

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

This article presents methods for generating, purifying, and quantifying Caenorhabditis elegans extracellular vesicles.

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular ...

Quantification of Insoluble Protein Aggregation in Caenorhabditis elegans during Aging with a Novel Data-Independent Acquisition Workflow

We and others have shown that the aging process results in a proteome-wide accumulation of insoluble proteins. Knocking down genes encoding the insoluble proteins over 40% of the time results in an extension of the lifespan in C. elegans, suggesting that many of these proteins are key determinants of the aging process. Isolation and quantitative identification of these insoluble proteins are crucial to understand key biological processes that occur during aging. Here, we present a modified and improved protocol that details how to extract and isolate the SDS-insoluble proteins (insolublome) from C. elegans more efficiently to streamline mass spectrometric workflows via a novel label-free quantitative proteomics analysis. This improved protocol utilizes a highly efficient sonicator for worm lysis that greatly increases efficiency for protein extraction and allows us to use significantly less starting material (approximately 3,000 worms) than in previous protocols (typically using at least 40,000 worms). Subsequent quantitative proteomic analysis of the insolublome was performed using data-dependent acquisition (DDA) for protein discovery and identification and data-independent acquisition (DIA) for comprehensive and more accurate protein quantification. Bioinformatic analysis of quantified proteins provides potential candidates that can be easily followed up with other molecular methods in C. elegans. With this workflow, we routinely identify more than 1000 proteins and quantify more than 500 proteins. This new protocol enables efficient compound screening with C. elegans. Here, we validated and applied this improved protocol to wild-type C. elegans N2-Bristol strain and confirmed that aged day-10 N2 worms showed greater accumulation of the insolublome than day-2 young worms.

Quantification of Insoluble Protein Aggregation in Caenorhabditis elegans during Aging with a Novel Data-Independent Acquisition Workflow

This novel workflow efficiently extracts and isolates SDS-insoluble proteins (insolublome) from Caenorhabditis elegans with minimal starting material for quantitative differential proteomic analysis. The protocol uses a comprehensive data-independent acquisition mass spectrometry analysis to quantify the insolublome and bioinformatic analysis to gain biological insights into aging mechanisms and pathologies.

We and others have shown that the aging process results in a proteome-wide accumulation of insoluble proteins. Knocking down genes encoding the insoluble ...

Small-Scale Extraction of Caenorhabditis elegans Genomic DNA

Genomic DNA extraction from single or a few Caenorhabditis elegans has many downstream applications, including PCR for genotyping lines, cloning, and sequencing. The traditional proteinase K-based methods for genomic DNA extraction from C. elegans take several hours. Commercial extraction kits that effectively break open the C. elegans cuticle and extract genomic DNA are limited. An easy, faster (~15 min), and cost-efficient method of extracting C. elegans genomic DNA that works well for classroom and research applications is reported here. This DNA extraction method is optimized to use single or a few late-larval (L4) or adult nematodes as starting material for obtaining a reliable template to perform PCR. The results indicate that the DNA quality is suitable for amplifying gene targets of different sizes by PCR, permitting genotyping of single or a few animals even at dilutions to one-fiftieth of the genomic DNA from a single adult per reaction. The reported protocols can be reliably used to quickly produce DNA template from a single or a small sample of C. elegans for PCR-based applications.

Small-Scale Extraction of Caenorhabditis elegans Genomic DNA

Presented here is a description of the straightforward and relatively rapid isolation of Caenorhabditis elegans genomic DNA from one or a few animals using a commercially available tissue kit. The resulting gDNA preparation is a suitable template for PCR.

Genomic DNA extraction from single or a few Caenorhabditis elegans has many downstream applications, including PCR for genotyping lines, cloning, and ...

Hand Dissection of Caenorhabditis elegans Intestines

Comprised of only 20 cells, the Caenorhabditis elegans intestine&#160;is the nexus of many life-supporting functions, including digestion, metabolism, aging, immunity, and environmental response. Critical interactions between the C. elegans host and its environment converge within the intestine, where gut microbiota concentrate. Therefore, the ability to isolate intestine tissue away from the rest of the worm is necessary to assess intestine-specific processes. This protocol describes a method for hand dissecting adult C. elegans intestines. The procedure can be performed in fluorescently labeled strains for ease or training purposes. Once the technique is perfected, intestines can be collected from unlabeled worms of any genotype. This microdissection approach allows for the simultaneous capture of host intestinal tissue and gut microbiota, a benefit to many microbiome studies. As such, downstream applications for the intestinal preparations generated by this protocol can include but are not limited to RNA isolation from intestinal cells and DNA isolation from captured microbiota. Overall, hand dissection of C. elegans intestines affords a simple and robust method to investigate critical aspects of intestine biology.

Hand Dissection of Caenorhabditis elegans Intestines

The present protocol describes a procedure for isolating intestines from adult Caenorhabditis elegans nematode worms by hand for input in genomics, proteomics, microbiome, or other assays.

Comprised of only 20 cells, the Caenorhabditis elegans intestine&#160;is the nexus of many life-supporting functions, including digestion, metabolism, ...

Single Cell Dissociation of Caenorhabditis elegans: A Method to Isolate Live Cells

Source: Germany, E. M., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/60145/isolation-specific-neuron-populations-from-roundworm-caenorhabditis">Isolation of Specific Neuron Populations from Roundworm Caenorhabditis elegans</a>. J. Vis. Exp. (2019).
This video describes a method to dissociate whole worms into a single-cell suspension suitable for FACS or immunoprecipitation of intact cells.

Source: Germany, E. M., et al. Isolation of Specific Neuron Populations from Roundworm Caenorhabditis elegans. J. Vis. Exp. (2019).
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Protein Extract Preparation and Co-immunoprecipitation from Caenorhabditis elegans

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Single Cell Dissociation of Caenorhabditis elegans: A Method to Isolate Live Cells