B.A-C is funded by the Spanish Ministry of Science and Innovation (Ram&#243;n y Cajal-RYC2018-024435-I), by the Autonomous Community of Madrid (Atracci&#243;n de Talento-2019T1/BMD-14057), and MICINN (PID2020-113270RA-I00) grants. R. A-P is funded by Autonomous Community of Madrid (Atracci&#243;n de Talento-2019T1/BMD-14057). E.M.S. is funded by the Max Planck Society, an Advanced Investigator award from the European Research Council (grant 743216), DFG CRC 1080: Molecular and Cellular Mechanisms of Neural Homeostasis, and DFG CRC 902: Molecular Principles of RNA-based Regulation. We thank D.C Dieterich and P. Landgraf for their technical advise and the synthesis of the DST-Alkyne. We thank E. Northrup, S. Zeissler, S. Gil Mast, and the animal facility of the MPI for Brain Research for their excellent support. We thank Sandra Goebbels for sharing the Nex-Cre mouse line. We thank Antonio G. Carroggio for his help with English editing. B.A-C. designed, conducted, and analyzed experiments. B.N-A, D.O.C, R.A-P, C. E., and S. t. D. conducted and analyzed experiments. B.A-C and E.M.S. designed experiments, and supervised the project, B.A-C wrote the paper. All authors edited the paper.

All experiments with animals were performed with permission from the local government offices in Germany (RP Darmstadt; protocols: V54-19c20/15-F122/14, V54-19c20/15-F126/1012) or Spain (Committee of Animal Experiments at the UCM and Environmental Counselling of the Comunidad de Madrid, protocol number: PROEX 005.0/21) and are compliant with the Max Planck Society rules and Spanish regulations and follow the EU guidelines for animal welfare.
1. In vivo metabolic labeling with ANL
<ol>
	<li>Drinking water:
	<ol>
		<li>Dissolve ANL and maltose in the regular drinking water of the mice. The recommended maltose concentration is 0.7% (wt/vol). The maximum amount of ANL added to the drinking water is 1% (wt/vol). Once the mixture is ready, sterilize it by filtration and store it at 4 &#176;C.</li>
		<li>Provide the mixture prepared in step 1.1 to the mice. Change bottles frequently (e.g., every 3 days) to avoid contaminations, and check them every day to look for possible contamination or clogging. Monitor the amount of drunk water daily by weighing it, to know mice&#39;s ANL intake. 
		NOTE: The maximum labeling period evaluated here is 3 weeks using an ANL concentration of 1% in the drinking water, which resulted in well-detectable labeling in the brain. Good labeling can also be achieved in 2 weeks. Neither shorter labeling times, nor longer time points, nor other ANL amounts have been studied by us using this administration method, which does not imply that the foregoing would not work. ANL incorporation rates can vary depending on the studied tissue or cell-type. The time of labeling can vary depending on the scientific question; for example, if proteomes were to be labeled, then labeling time should be calculated in function of the average half-lives of the proteins in the studied tissue. If the interest is only to identify newly synthesized proteins, the times can be shortened. The labeling periods and ANL dosages have to be empirically tested for each experimental condition and cell-type of interest.</li>
	</ol>
	</li>
	<li>Intraperitoneal administration:
	<ol>
		<li>Dissolve ANL in physiological NaCl solution to 400 mM, phosphate saline buffer (PBS), or water.</li>
		<li>Make sure that the osmolarity of the solution is within the physiological range. Here, 10 mL/kg body weight of ANL solution is administered to the mice. 
		&#8203;NOTE: Good labeling in the excitatory neurons of the brain is successfully obtained by one daily intraperitoneal injection (IP) with 400 mM ANL for 1 week. Neither lower ANL concentrations nor other labeling periods by IP injections have been tested in this study, which does not imply they would not work. ANL is supplied as hydrochloride by some companies. In this case, the pH of the solutions must be adjusted to the adequate pH (depending on the administration route). ANL can also be synthesized following the method published by Link et al.11 with some modifications5. ANL labeling can be performed using other ANL concentrations, timespans, and administration routes. For tissues/cell-types with low metabolic rates, a low content methionine diet can be used, always starting 1 week before ANL administration. When 400 mM ANL is used, it can precipitate while stored at 4 &#176;C; heating up to 37 &#176;C brings ANL back into the solution. Let it reach room temperature before injection. In most world regions, ANL administration to mice is an animal experiment and needs to be approved by the pertinent authorities.</li>
	</ol>
	</li>
</ol>
2. Tissue harvesting, lysis, and protein extraction
<ol>
	<li>Tissue dissection: Dissect the area of tissue containing the cell-type of interest and note the weight of the collected piece of tissue. 
	NOTE: Samples can be stored at -80 &#176;C for several months (stop point). In the present protocol, mice cortex and hippocampus were dissected.</li>
	<li>Tissue lysis: Homogenize the tissue at room temperature by adding a volume 12-15 times the wet weight of the tissue of the lysis buffer (PBS pH 7.4, 1% wt/vol SDS, 1% (vol/vol) Triton X-100, Benzonase (1:1000 vol/vol), a protease inhibitor (PI) (EDTA free 1:4000)). For example, for a 20 mg tissue piece, add 240-300 &#956;L of lysis buffer. Triturate the tissue until it is homogenized. 
	NOTE: Samples can be stored at -80 &#176;C for several months (stop point). For direct homogenization in 1.5 mL tubes, the use of a handheld, battery-operated homogenizer is recommended, especially when small pieces of tissue are processed. For bigger pieces, a Dounce homogenizer can be used. To every buffer where protease inhibitors (PI) are included, they should be added immediately before use and not earlier.</li>
	<li>Protein denaturation: Heat the homogenate to 75 &#176;C for 15 min and centrifuge at 17,000 x g for 15 min at 10 &#176;C. Transfer the supernatant to a new tube.</li>
	<li>Protein content measurement: Measure the protein concentration of each sample, and adjust all the samples to be compared to the same protein concentration; adjust the concentration by adding lysis buffer. An optimal concentration range is between 2-4 &#956;g/&#956;L. 
	NOTE: Samples can be stored at -80 &#176;C for several months (stop point).</li>
	<li>Alkylation
	<ol>
		<li>Dilute the homogenized samples by 2-3 times in PBS (pH 7.8) + PI (EDTA free 1:4000).</li>
		<li>Add iodoacetamide (IAA) to a final concentration of 20 mM (stock solution 500 mM).</li>
		<li>Leave the samples for 1-2 h at 20 &#176;C in the dark. Do steps 2.5.2-2.5.3 twice. 
		NOTE: For some tissues, if the non-specific click in the negative control remains high, it might be necessary to perform a reduction step prior to alkylation12. Prepare the IAA stock freshly just before adding it to the samples. Samples can be stored at -80 &#176;C for several months (stop point).</li>
	</ol>
	</li>
	<li>Buffer exchange: Equilibrate buffer exchange columns using the click chemistry exchange buffer (PBS pH 7.8, 0.04% (wt/vol) SDS, 0.08% (vol/vol) Triton X-100 and PI (1:4000)). Follow the manufacturer&#39;s instructions. Exchange all alkylated samples. 
	NOTE: Eluted samples can be stored at -80 &#176;C for several months (stop point). In this step, the IAA is eliminated, and the buffer is exchanged by the click chemistry buffer. Protein can be measured again after the buffer exchange step to make sure that the proteins were not lost during the buffer exchange process. Depending on the type of columns used for buffer exchange, protein can be massively lost. Use the recommended columns to avoid protein loss. For sample storage, the preparation of aliquots is recommended.</li>
	<li>Click reaction to evaluate ANL incorporation in the experiment
	<ol>
		<li>Take 40 &#956;L of the eluted samples in step 2.6 and add PBS (pH 7.8) to a final volume of 120 &#181;L.</li>
		<li>To set up the click chemistry reaction, add the following reagents in the given order and vortex for 20 s following each addition: 1.5 &#181;L of triazole ligand (stock 40 mM), 1.5 &#181;L of biotin alkyne (stock 5 mM) and 1 &#181;L of Cu(I) bromide (stock 10 mg/mL, dissolved in DMSO by careful pipetting up and down, do not vortex). Add the reagents as fast as possible.</li>
		<li>Incubate the samples overnight in the dark at 4 &#176;C with continuous rotation.</li>
		<li>Centrifuge the samples at 4 &#176;C for 5 min at 17,000 x g. A slightly turquoise pellet will be visible. Transfer the supernatant to a new tube. 
		NOTE: Prepare Cu (I) bromide stock immediately before use to avoid copper oxidation. Keeping the ratio of volumes between the lysed sample and PBS constant between samples is essential to ensure the same final detergent concentration in each tube. To speed up the assembly of the click chemistry reaction, a table vortexer with racks for tubes is recommended. Store the clicked samples at -80 &#176;C for several months, or at -20 &#176;C for a couple of weeks until further processing (stop point).</li>
	</ol>
	</li>
	<li>Western blot analysis to evaluate ANL incorporation in the experiment
	<ol>
		<li>Run an SDS-PAGE with 20-40 &#956;L of the supernatant from the click chemistry reaction (step 2.7). Use 12% acrylamide gels, run in 1% SDS, 250 mM Tris-HCl, 2 mM glycine. Run until the front is out of the gel.</li>
		<li>Transfer the proteins to a nitrocellulose or PVDF membrane13.</li>
		<li>Incubate the membrane overnight at 4 &#176;C with GFP antibody (1:500, GFP protein is co-translated with MetRS*5), and biotin antibody (1:1000) for clicked alkyne detection, which is conjugated to biotin.</li>
		<li>Wash the primary antibody 2 times for 10 min and add the secondary antibody for 1.5 h.</li>
		<li>Wash the secondary antibody 2 times for 10 min and scan (if using fluorophore-conjugated antibodies) or expose to films (if using horseradish peroxidase-conjugated antibodies).</li>
		<li>Quantify the biotin signal using ImageJ or similar software, evaluate the general labeling of each sample of the experiment, and detect possible outliers. Evaluate the signal-to-noise ratio (ANL sample labeling to background from the negative controls) of the experiment and detect possible animals which failed to take up/incorporate ANL. 
		NOTE: For samples with high ANL incorporation, protein purification is theoretically possible using non-cleavable alkynes like the one used in this section for the evaluation of ANL incorporation. Using brain samples, the background obtained in the negative controls by MS after protein purification using non-cleavable alkynes is too high14. Therefore, only the easier-to-handle, non-cleavable alkynes were used for a first general sample and experiment evaluation. Regardless of the type of alkyne intended for protein purification, it is advisable to perform the steps for alkyne dosage optimization described in the following section (step 2.9).</li>
	</ol>
	</li>
	<li>Click reaction for cleavable alkyne dosage optimization
	<ol>
		<li>Prepare four tubes with 40 &#956;L of one representative sample (obtained in step 2.6 and evaluated in step 2.8) and add PBS (pH 7.8) to a final volume of 120 &#956;L.</li>
		<li>Set up the click chemistry reaction as explained in step 2.7 but this time, add four different amounts of the DST-alkyne. For said alkyne, testing 5 &#956;M, 15 &#956;M, 30 &#956;M, and 60 &#956;M is recommended.</li>
		<li>Repeat step 2.8 to decide which is the most suitable alkyne dosage for the specific experimental question under study, based on the highest labeling efficiency with the lowest background signal. 
		NOTE: Before subjecting the remaining samples to a click reaction, the optimal amount of any alkyne needs to be determined. There are several options of commercially available alkynes that can be used. They differ in the way of cleavage (e.g., light or reducing agents). Copper-free click chemistry using strain-promoted reagents (e.g., DBCO reagents) is also possible. Tiny changes in the amount of either alkynes or DBCO reagents often increase the signal in the negative control (background) significantly. Here, the protein purification using an alkyne with a disulfide bridge cleavable by reducing agents (Disulfide biotin alkyne or DST-alkyne15) is described. When using the DST-alkyne, for running the SDS-PAGE (step 8.1), avoid loading buffers with strong reducing agents such as DTT or &#946;-Mercaptoethanol to prevent alkyne cleavage. In this study, heating at 72 &#176;C, for 5 min, using 5 mM of N-ethylmaleimide (NEM) in the loading buffer does not affect DST-alkyne stability. Step 2.9.2 can be repeated, testing more fine-grained dosages in the range of the best one observed.</li>
	</ol>
	</li>
	<li>Preparative click reaction for protein purification
	<ol>
		<li>Click all the samples obtained in step 2.6 with the optimal alkyne dosage determined in step 2.9 and analyze a fraction by SDS-PAGE and Western blot (step 2.8). 
		&#8203;NOTE: Make sure that the pipettes are properly calibrated to ensure sample upscaling accuracy. Clicked samples can be stored at -80 &#176;C for several months (stop point).</li>
	</ol>
	</li>
</ol>
3. Protein purification
<ol>
	<li>Buffer exchange
	<ol>
		<li>Subject all clicked samples to a buffer exchange procedure as described in step 2.6, but use Neutravidin binding buffer as equilibration buffer (PBS pH 7.4, 0.15% (wt/vol) SDS, 1% (vol/vol) Triton X-100 and PI (1:2000)). 
		NOTE: Eluted samples can be stored at -80 &#176;C for several months (stop point). In this step, the non-clicked free alkyne is eliminated, and the buffer is exchanged by the Neutravidin binding buffer.</li>
	</ol>
	</li>
	<li>Binding to Neutravidin beads
	<ol>
		<li>Wash the Neutravidin high-capacity beads with the Neutravidin binding buffer (see step 3.1); mix the beads with the buffer and centrifuge the mixture for 5 min at 3000 x g, and discard the supernatant. Repeat three times.</li>
		<li>Prepare a 1:1 slurry of Neutravidin beads by adding the same volume of dry beads and Neutravidin binding buffer.</li>
		<li>Set aside 20-40 &#956;L of each sample as pre-purified lysate and store it at -20 &#176;C.</li>
		<li>Measure protein concentration (see step 2.4), and mix 100 &#956;g of protein with 4 &#956;L of the bead slurry obtained in step 3.2.2.</li>
		<li>Incubate the mixture overnight at 4 &#176;C with continuous rotation, enabling labeled proteins to bind to the beads. 
		NOTE: As for the click chemistry reaction, the basic affinity purification reaction described in step 3.2.4 can be upscaled. For example, for purification of proteins from the excitatory neurons of the hippocampus, 1 mg of protein lysate is mixed with 40 &#956;L of Neutravidin beads (1:1 slurry). The amount of Neutravidin beads can be scaled up or down according to the amount of labeled proteins per mg of total protein, which will depend on the chosen cell-type and labeling period and needs to be determined empirically.</li>
	</ol>
	</li>
	<li>Neutravidin beads washes and protein elution
	<ol>
		<li>Collect the supernatants by centrifugation (see step 3.2.1). Set a 20-40 &#956;l aliquot of each supernatant aside for later analysis and freeze the rest at -80 &#176;C.</li>
		<li>Wash the beads with chilled Neutravidin washing buffer 1 (PBS pH 7.4, 0.2% (wt/vol) SDS, 1% (vol/vol) Triton X-100 and PI (1:2000)) by adding the buffer, sedimenting the beads, and discarding the supernatant. Repeat this step three times.</li>
		<li>Then, add the same buffer and incubate the beads for 10 min under continuous rotation at 4 &#176;C before discarding the supernatant. Repeat this step three times.</li>
		<li>Wash the beads as explained in steps 3.3.2-3.3.3 but using Neutravidin washing buffer 2 (1x PBS, pH 7.4, and PI 1:2000).</li>
		<li>Wash the beads as explained the step 3.3.2-3.3.3 but using Neutravidin washing buffer 3 (50 mM ammonium bicarbonate and PI 1:2000).</li>
		<li>Elute the clicked proteins by incubating the beads for 30 min at 20 &#176;C with a volume of Neutravidin elution buffer (5% (vol/vol) &#946;-mercaptoethanol and 0.03% (wt/vol) SDS) corresponding to one volume of the dry beads used.</li>
		<li>Maintain the beads in suspension during elution by continuous agitation (1000 rpm) in a thermoblock shaker. Elute twice and combine both eluates. 
		NOTE: The centrifugation set up for separation of the solid fraction of the slurry (beads) and the aqueous phase (supernatant), as explained in step 3.2.1, applies to steps 3.3.1-3.3.7. The SDS amount can be increased if protein elution is not complete. However, with amounts above 0.08%-0.1% of SDS, proteins non-specifically bound to the beads start to be eluted. &#946;-mercaptoethanol is volatile and toxic; the use of a hood for steps 3.3.6-3.3.7 is advisable. The samples collected in steps 3.2.3 and 3.3.1 should be run by SDS-PAGE and evaluated by Western blot (see step 2.8) for evaluating the efficiency of the affinity purification (step 3.3).</li>
	</ol>
	</li>
	<li>Evaluation of eluted proteins
	<ol>
		<li>Load 1/3 of the eluted samples to an SDS-PAGE (see step 2.8.1).</li>
		<li>Stain the gel to visualize proteins using a high sensitivity method, such as silver staining or a fluorescent method.</li>
		<li>If the samples have the expected quality, meaning that there is 3-4-fold more protein in the ANL labeled samples compared to the negative control, the eluted proteins can be identified by routine mass spectrometry methods like the one explained in reference5.</li>
	</ol>
	</li>
</ol>

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K., Clavarino, G., Ceppi, M., Pierre, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SUnSET,+a+nonradioactive+method+to+monitor+protein+synthesis.">SUnSET, a nonradioactive method to monitor protein synthesis.</a> Nature Methods. 6 (4), 275-277 (2009).</li><li>Roux, K. J., Kim, D. I., Burke, B., May, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BioID:+A+screen+for+protein-protein+interactions.">BioID: A screen for protein-protein interactions.</a> Current Protocols in Protein Science. 91, 11-15 (2018).</li></ol>

The authors declare no competing financial interests.

The critical aspects of the protocol are; the inclusion of negative controls, having enough biological replicates, ANL administration route, amount, and duration, alkylation of the samples, alkyne concentration, and &#946;-mercaptoethanol elimination when using DST-alkyne.
It is key to include negative control samples proceeding from ANL-labeled animals without Cre driver and therefore no MetRS* expression. These samples must be subjected to every step described in the protocol in parallel to ...

Aberrant protein homeostasis is caused by an imbalance in protein synthesis and degradation. Several diseases are related to alterations in protein homeostasis. The hallmark of some diseases is the presence of aggregates in different subcellular locations and brain areas. Protein homeostasis is not only important in disease but also plays a crucial role in normal organ and cellular function1. For example, protein synthesis is necessary for many forms of neuronal plasticity2,3, as determined by the use of chemical inhibitors that block protein synthesis4. However, it is neither clear in which cell-types the proteome is altered to support learning and memory, nor is it understood which specific proteins in each cell-type increase or decrease in their synthesis or degradation. Thus, a comprehensive study of protein homeostasis requires the capability to differentiate proteomes coming from specific cell-types. Indeed, the identification of cell-type-specific proteomes to study cellular processes occurring in a multicellular environment has been an important hurdle in proteomics. For this reason, we developed a technique using MetRS* expression combined with bio-orthogonal methods that has proven to be an effective way to identify and purify cell-type specific proteomes, filling this gap5,6,7.
The expression of a mutant MetRS* (MetRS L274G) allows for the loading of the non-canonical methionine analog ANL into the corresponding tRNA8,9 and its subsequent incorporation&#160;into proteins. When MetRS* expression is regulated by a cell-type-specific promoter, the non-canonical amino acid will be incorporated into the proteins in a cell-selective manner. Once ANL is incorporated in the proteins, it can be selectively functionalized by click-chemistry and subsequently either visualized by imaging (FUNCAT) or by Western Immunoblot (BONCAT). Alternatively, proteins can be selectively purified and identified by mass spectrometry (MS). Using this technology, we created a mouse line expressing the MetRS* protein under the control of the Cre recombinase. Considering the increasing number of available Cre-mouse lines, the MetRS* system can be used in any field to study any cell-type from any tissue for which there is an existing Cre-line. Protein labeling with ANL is possible in vitro or in vivo, and does not alter mouse behavior or protein integrity6. Labeling timespan can be adapted to the scientific question of each researcher, labeling newly synthesized proteins (shorter labeling times) or entire proteomes (longer labeling times). The use of this technique is limited by the number of cells of the type that the researcher is willing to study; hence protein isolation from cell-types with low numbers or low metabolic rates is not possible by this method. The goal of the presented method is to identify cell-type-specific proteins/proteomes labeled in vivo. In this protocol, we describe how to label cell-type-specific proteomes with ANL in live mice and purify the labeled proteins. After purification, proteins can be identified by routine mass spectrometry protocols5,10. The reduction of sample complexity achieved in this method by the selective purification of proteins from specific cellular populations allows the experimenter to detect subtle changes in proteomes, for example, in response to environmental changes. Purification of the labeled proteins can be achieved in ~10 days, not including the MS analysis or the labeling period. Here, we describe two methods for ANL administration to MetRS* expressing mice, namely (1) adding the amino acid in the drinking water, and (2) introducing ANL by intraperitoneal injections. Regardless of the method chosen for ANL administration, the isolation and purification steps are the same (from step 2 on).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>12% Acrylamide gels</td>
			<td>GenScript</td>
			<td>SurePAGE, Bis-Tris, 10 x 8, 12%</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>Sigma</td>
			<td>M6250</td>
			<td>Toxic; use a lab coat, gloves and a fume hood.</td>
		</tr>
		<tr>
			<td>Ammonium bicarbonate</td>
			<td>Sigma</td>
			<td>9830</td>
			<td>Toxic; use a lab coat, gloves and a fume hood</td>
		</tr>
		<tr>
			<td>ANL</td>
			<td></td>
			<td></td>
			<td>Synthesized as described previously for AHA (see references 5 and 11)</td>
		</tr>
		<tr>
			<td>ANL-HCl</td>
			<td>IrishBotech</td>
			<td>HAA1625.0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Benzonase</td>
			<td>Sigma</td>
			<td>E1014</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin alkyne</td>
			<td>Thermo,</td>
			<td>B10185</td>
			<td></td>
		</tr>
		<tr>
			<td>Chicken antibody anti-GFP</td>
			<td>Aves</td>
			<td>1020</td>
			<td></td>
		</tr>
		<tr>
			<td>Complete EDTA-free protease inhibitor</td>
			<td>Roche</td>
			<td>4693132001</td>
			<td>Toxic; use a lab coat and gloves.</td>
		</tr>
		<tr>
			<td>Copper (I) bromide</td>
			<td>Sigma</td>
			<td>254185</td>
			<td>99.999% (wt/wt)</td>
		</tr>
		<tr>
			<td>Disulfide tag (DST)-alkyne</td>
			<td></td>
			<td></td>
			<td>Synthesized as reported in reference number 15, in which it is referred to as probe 20</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>276855</td>
			<td></td>
		</tr>
		<tr>
			<td>Filters</td>
			<td>Merk</td>
			<td>SCGP00525</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodo acetamide (IAA)</td>
			<td>Sigma</td>
			<td>I1149</td>
			<td></td>
		</tr>
		<tr>
			<td>IR anti chicken 800</td>
			<td>LI-COR</td>
			<td>IRDye 800CW</td>
			<td>Donkey anti-Chicken Secondary Antibody</td>
		</tr>
		<tr>
			<td>IR anti rabit 680</td>
			<td>LI-COR</td>
			<td>IRDye 680RD</td>
			<td>Goat anti-Rabbit IgG Secondary Antibody</td>
		</tr>
		<tr>
			<td>Maltose</td>
			<td>Sigma</td>
			<td>M9171</td>
			<td></td>
		</tr>
		<tr>
			<td>Manual Mixer</td>
			<td>BioSpec Products</td>
			<td>1083</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>N-ethylmaleimide</td>
			<td>Sigma</td>
			<td>4259</td>
			<td>Toxic; use a lab coat, gloves and a fume hood.</td>
		</tr>
		<tr>
			<td>NeutrAvidin beads</td>
			<td>Pierce</td>
			<td>29200</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrocellulose membrane</td>
			<td>Bio-rad</td>
			<td>1620112</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS 1X</td>
			<td>Thermo</td>
			<td>J62036.K2</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS 1X pH 7.8</td>
			<td></td>
			<td></td>
			<td>Preparation described in reference number 5</td>
		</tr>
		<tr>
			<td>PD SpinTrap G-25 columns</td>
			<td>GE Healthcare</td>
			<td></td>
			<td>Buffer exchange</td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit</td>
			<td>Thermo,</td>
			<td>23225</td>
			<td>Reagents in the Pierce BCA Protein Assay Kit are toxic to aquatic life.</td>
		</tr>
		<tr>
			<td>Polyclonal rabbit anti-biotin antibody</td>
			<td>Cell Signaling</td>
			<td>5597</td>
			<td></td>
		</tr>
		<tr>
			<td>PVDF membrane</td>
			<td>Millipore</td>
			<td>IPVH00010</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS 10%</td>
			<td>Sigma</td>
			<td>71736</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS-PAGE&#160; Running buffer MOPS</td>
			<td>GenScript</td>
			<td>M00138</td>
			<td></td>
		</tr>
		<tr>
			<td>SYPRO Ruby stain</td>
			<td>Sigma</td>
			<td>S4942</td>
			<td></td>
		</tr>
		<tr>
			<td>Table automatic Vortexer</td>
			<td>Eppendorf</td>
			<td>Mixmate</td>
			<td></td>
		</tr>
		<tr>
			<td>Triazole ligand</td>
			<td>Sigma</td>
			<td>678937</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T9284</td>
			<td></td>
		</tr>
		<tr>
			<td>Water</td>
			<td>Sigma</td>
			<td>W4502</td>
			<td>Molecular biology grade</td>
		</tr>
	</tbody>
</table>

cell-type specific protein purification and identification from complex tissues using a mutant methionine trna synthetase mouse line

Understanding protein homeostasis in vivo is key to knowing how the cells work in both physiological and disease conditions. The present protocol describes in vivo labeling and subsequent purification of newly synthesized proteins using an engineered mouse line to direct protein labeling to specific cellular populations. It is an inducible line by Cre recombinase expression of L274G-Methionine tRNA synthetase (MetRS*), enabling azidonorleucine (ANL) incorporation to the proteins, which otherwise will not occur. Using the method described here, it is possible to purify cell-type-specific proteomes labeled in vivo and detect subtle changes in protein content due to sample complexity reduction.

Following the described protocol (summarized in Figure 1), ANL was administered to mice either by daily intraperitoneal injections (400 mM ANL 10 mL/kg, Nex-Cre::MetRS*) for 7 days, or via drinking water (0.7% Maltose, 1% ANL, CamkII-Cre::MetRS*) for 21 days. After labeling, the corresponding brain areas were dissected, lysed, alkylated, and clicked. Click reactions were analyzed by SDS-PAGE and Western Immunoblot. Representative images of the experiments are shown ...

Watch this Scientific Journal Video about Cell-Type Specific Protein Purification and Identification from Complex Tissues Using a Mutant Methionine tRNA Synthetase Mouse Line at JoVE.com

Cell-Type Specific Protein Purification and Identification from Complex Tissues Using a Mutant Methionine tRNA Synthetase Mouse Line

This protocol describes how to perform cell-type-specific protein labeling with azidonorleucine (ANL) using a mouse line expressing a mutant L274G-Methionine tRNA synthetase (MetRS*) and the necessary steps for labeled cell-type-specific proteins isolation. We outline two possible ANL administration routes in live mice by (1) drinking water and (2) intraperitoneal injections.

Understanding protein homeostasis in vivo is key to knowing how the cells work in both physiological and disease conditions. The present protocol ...

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2.7K Views.  Synaptic Plasticity Department. This protocol describes how to perform cell-type-specific protein labeling with azidonorleucine (ANL) using a mouse line expressing a mutant L274G-Methionine tRNA synthetase (MetRS*) and the necessary steps for labeled cell-type-specific proteins isolation. We outline two possible ANL administration routes in live mice by (1) drinking water and (2) intraperitoneal injections. 

Video: Cell-Type Specific Protein Purification and Identification from Complex Tissues Using a Mutant Methionine tRNA Synthetase Mouse Line

Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient

Synaptoneurosomes (SNs) are obtained after homogenization and fractionation of mouse brain cortex. They are resealed vesicles or isolated terminals that break away from axon terminals when the cortical tissue is homogenized. The SNs retain pre- and postsynaptic characteristics, which makes them useful in the study of synaptic transmission. They retain the molecular machinery used in neuronal signaling and are capable of uptake, storage, and release of neurotransmitters.
The production and isolation of active SNs can be problematic using medias like Ficoll, which can be cytotoxic and require extended centrifugation due to high density, and filtration and centrifugation methods, which can result in low activity due to mechanical damage of the SNs. However, the use of discontinuous Percoll-sucrose density gradients to isolate SNs provides a rapid method to produce good yields of translationally active SNs. The Percoll-sucrose gradient method is quick and gentle as it employs isotonic conditions, has fewer and shorter centrifugation spins and avoids centrifugation steps that pellet SNs and cause mechanical damage.

A method to prepare translationally active, intact synaptoneurosomes (SNs) from mouse brain cortex is described. The method uses a discontinuous Percoll-sucrose density gradient allowing for the quick preparation of active SNs.

Synaptoneurosomes (SNs) are obtained after homogenization and fractionation of mouse brain cortex. They are resealed vesicles or isolated terminals that ...

Isolating LacZ-expressing Cells from Mouse Inner Ear Tissues using Flow Cytometry

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues presents a unique challenge because of the paucity of cells of interest and few transgenic reporter mouse models. Here, we describe a protocol using fluorescence-conjugated probes to selectively label LacZ-positive cells from the neonatal cochleae.
The most common underlying pathology of sensorineural hearing loss is the irreversible damage and loss of cochlear sensory hair cells, which are required to transduce sound waves to neural impulses. Recent evidence suggests that the murine auditory and vestibular organs harbor stem/progenitor cells that may have regenerative potential1,2. These findings warrant further investigation, including identifying specific cell types with stem/progenitor cell characteristics. The Wnt signaling pathway has been demonstrated to play a critical role in maintaining stem/progenitor cell populations in several organ systems3-7. We have recently identified Wnt-responsive Axin2-expressing cells in the neonatal cochlea, but their function is largely unknown8.
To better understand the behavior of these Wnt-responsive cells in vitro, we have developed a method of isolating Axin2-expressing cells from cochleae of Axin2-LacZ reporter mice9. Using flow cytometry to isolate Axin2-LacZ positive cells from the neonatal cochleae, we could in turn execute a variety of experiments on live cells to interrogate their behavior as stem/progenitor cells. Here, we describe in detail the steps for the microdissection of neonatal cochlea, dissociation of these tissues, labeling of the LacZ-positive cells using a fluorogenic substrate, and cell sorting. Techniques for dissociating cochleae into single cells and isolating cochlear cells via flow cytometry have been described2,10-12. We have made modifications to these techniques to establish a novel protocol to isolate LacZ-expressing cells from the neonatal cochlea.

Flow cytometry is a powerful tool allowing for the isolation and study of specific cell populations. This protocol describes steps for isolating LacZ-expressing cells from cochlear tissues from neonatal transgenic mice. Dissociated cochlear cells were labeled using fluorescent-conjugated substrates of &#946;-galactosidase prior to separation via flow cytometry.

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues ...

Isolation and Quantification of Botulinum Neurotoxin From Complex Matrices Using the BoTest Matrix Assays

Accurate detection and quantification of botulinum neurotoxin (BoNT) in complex matrices is required for pharmaceutical, environmental, and food sample testing. Rapid BoNT testing of foodstuffs is needed during outbreak forensics, patient diagnosis, and food safety testing while accurate potency testing is required for BoNT-based drug product manufacturing and patient safety. The widely used mouse bioassay for BoNT testing is highly sensitive but lacks the precision and throughput needed for rapid and routine BoNT testing. Furthermore, the bioassay&#39;s use of animals has resulted in calls by drug product regulatory authorities and animal-rights proponents in the US and abroad to replace the mouse bioassay for BoNT testing. Several in vitro replacement assays have been developed that work well with purified BoNT in simple buffers, but most have not been shown to be applicable to testing in highly complex matrices. Here, a protocol for the detection of BoNT in complex matrices using the BoTest Matrix assays is presented. The assay consists of three parts: The first part involves preparation of the samples for testing, the second part is an immunoprecipitation step using anti-BoNT antibody-coated paramagnetic beads to purify BoNT from the matrix, and the third part quantifies the isolated BoNT&#39;s proteolytic activity using a fluorogenic reporter. The protocol is written for high throughput testing in 96-well plates using both liquid and solid matrices and requires about 2 hr of manual preparation with total assay times of 4-26 hr depending on the sample type, toxin load, and desired sensitivity. Data are presented for BoNT/A testing with phosphate-buffered saline, a drug product, culture supernatant, 2% milk, and fresh tomatoes and includes discussion of critical parameters for assay success.

The BoTest Matrix botulinum neurotoxin (BoNT) detection assays rapidly purify and quantify BoNT from a range of sample matrices.&#160;Here, we present a protocol for the detection and quantification of BoNT from both solid and liquid matrices and demonstrate the assay with BOTOX, tomatoes, and milk.

Accurate detection and quantification of botulinum neurotoxin (BoNT) in complex matrices is required for pharmaceutical, environmental, and food sample ...

Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant cell type in the brain, neurons are the most widely studied of these cell types given their direct role in impacting behaviors. Other cell types in the brain also impact neuronal function and behavior via the signaling molecules they produce. Neuroscientists must understand the interactions between the cell types in the brain to better understand how these interactions impact neural function and disease. To date, the most common method of analyzing protein or gene expression utilizes the homogenization of whole tissue samples, usually with blood, and without regard for cell type. This approach is an informative approach for examining general changes in gene or protein expression that may influence neural function and behavior; however, this method of analysis does not lend itself to a greater understanding of cell-type-specific gene expression and the effect of cell-to-cell communication on neural function. Analysis of behavioral epigenetics has been an area of growing focus which examines how modifications of the deoxyribonucleic acid (DNA) structure impact long-term gene expression and behavior; however, this information may only be relevant if analyzed in a cell-type-specific manner given the differential lineage and thus epigenetic markers that may be present on certain genes of individual neural cell types. The Fluorescence Activated Cell Sorting (FACS) technique described below provides a simple and effective way to isolate individual neural cells for the subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA. This technique can also be modified to isolate more specific neural cell types in the brain for subsequent cell-type-specific analysis.

The goal of this protocol is to use the fluorescence activated cell sorting (FACS) technique to sort specific types of neural cells for subsequent analysis of cell-type-specific gene expression, epigenetic markers, and or protein expression.

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant ...

Purification of Mouse Brain Vessels

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and immune cells between the brain and the blood. Moreover, the huge neuronal metabolic demand requires a moment-to-moment regulation of blood flow. Notably, abnormalities of these regulations are etiological hallmarks of most brain pathologies; including glioblastoma, stroke, edema, epilepsy, degenerative diseases (ex: Parkinson&#8217;s disease, Alzheimer&#8217;s disease), brain tumors, as well as inflammatory conditions such as multiple sclerosis, meningitis and sepsis-induced brain dysfunctions. Thus, understanding the signaling events modulating the cerebrovascular physiology is a major challenge. Much insight into the cellular and molecular properties of the various cell types that compose the cerebrovascular system can be gained from primary culture or cell sorting from freshly dissociated brain tissue. However, properties such as cell polarity, morphology and intercellular relationships are not maintained in such preparations. The protocol that we describe here is designed to purify brain vessel fragments, whilst maintaining structural integrity. We show that isolated vessels consist of endothelial cells sealed by tight junctions that are surrounded by a continuous basal lamina. Pericytes, smooth muscle cells as well as the perivascular astrocyte endfeet membranes remain attached to the endothelial layer. Finally, we describe how to perform immunostaining experiments on purified brain vessels.

We describe a protocol allowing the purification of the mouse brain's vascular compartment. Isolated brain vessels include endothelial cells linked by tight junctions and surrounded by a continuous basal lamina, pericytes, vascular smooth muscle cells, as well as perivascular astroglial membranes.

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and ...

External Excitation of Neurons Using Electric and Magnetic Fields in One- and Two-dimensional Cultures

A neuron will fire an action potential when its membrane potential exceeds a certain threshold. In typical activity of the brain, this occurs as a result of chemical inputs to its synapses. However, neurons can also be excited by an imposed electric field. In particular, recent clinical applications activate neurons by creating an electric field externally. It is therefore of interest to investigate how the neuron responds to the external field and what causes the action potential. Fortunately, precise and controlled application of an external electric field is possible for embryonic neuronal cells that are excised, dissociated and grown in cultures. This allows the investigation of these questions in a highly reproducible system.
In this paper some of the techniques used for controlled application of external electric field on neuronal cultures are reviewed. The networks can be either one dimensional, i.e. patterned in linear forms or allowed to grow on the whole plane of the substrate, and thus two dimensional. Furthermore, the excitation can be created by the direct application of electric field via electrodes immersed in the fluid (bath electrodes) or by inducing the electric field using the remote creation of magnetic pulses.

Neuronal cultures are a good model for studying emerging brain stimulation techniques via their effect on single neurons or a population of neurons. Presented here are different methods for stimulation of patterned neuronal cultures by an electric field produced directly by bath electrodes or induced by a time-varying magnetic field.

A neuron will fire an action potential when its membrane potential exceeds a certain threshold. In typical activity of the brain, this occurs as a result ...

Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral tissues. The hypothalamus in the central nervous system (CNS) is the control center for energy and insulin signal response regulation. Chronic inflammation in peripheral tissues and imbalances of certain chemokines (such as CCL5, TNF&#945;, and IL-6) contribute to diabetes and obesity. However, the functional mechanism(s) connecting chemokines and hypothalamic insulin signal regulation still remain unclear.
In vitro primary neuron culture models are convenient and simple models which can be used to investigate insulin signal regulation in hypothalamic neurons. In this study, we introduced exogeneous GLUT4 protein conjugated with GFP (GFP-GLUT4) into primary hypothalamic neurons to track GLUT4 membrane translocation upon insulin stimulation. Time-lapse images of GFP-GLUT4 protein trafficking were recorded by deconvolution microscopy, which allowed users to generate high-speed, high-resolution images without damaging the neurons significantly while conducting the experiment. The contribution of CCR5 in insulin regulated GLUT4 translocation was observed in CCR5 deficient hypothalamic neurons, which were isolated and cultured from CCR5 knockout mice. Our results demonstrated that the GLUT4 membrane translocation efficiency was reduced in CCR5 deficient hypothalamic neurons after insulin stimulation.

This protocol describes a technique for observation of real-time Green Fluorescence Protein (GFP) tagged Glucose Transporter 4 (GLUT4) protein trafficking upon insulin stimulation and characterization of the biological role of CCR5 in the insulin&#8211;GLUT4 signaling pathway with Deconvolution Microscopy.

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral ...

Rapid and Specific Immunomagnetic Isolation of Mouse Primary Oligodendrocytes

The efficient and robust isolation and culture of primary oligodendrocytes (OLs) is a valuable tool for the in vitro study of the development of oligodendroglia as well as the biology of demyelinating diseases such as multiple sclerosis and Pelizaeus-Merzbacher-like disease (PMLD). Here, we present a simple and efficient selection method for the immunomagnetic isolation of stage three O4+ preoligodendrocytes cells from neonatal mice pups. Since immature OL constitute more than 80% of the rodent-brain white matter at postnatal day 7 (P7) this isolation method not only ensures high cellular yield, but also the specific isolation of OLs already committed to the oligodendroglial lineage, decreasing the possibility of isolating contaminating cells such as astrocytes and other cells from the mouse brain. This method is a modification of the techniques reported previously, and provides oligodendrocyte preparation purity above 80% in about 4 h.

We describe the immunomagnetic isolation of primary mouse oligodendrocytes, which allows the rapid and specific isolation of the cells for in vitro culture.

The efficient and robust isolation and culture of primary oligodendrocytes (OLs) is a valuable tool for the in vitro study of the development of ...

Quantifying the Heterogeneous Distribution of a Synaptic Protein in the Mouse Brain Using Immunofluorescence

The presence, absence, or levels of specific synaptic proteins can severely influence synaptic transmission. In addition to elucidating the function of a protein, it is vital to also determine its distribution. Here, we describe a protocol employing immunofluorescence, confocal microscopy, and computer-based analysis to determine the distribution of the synaptic protein Mover (also called TPRGL or SVAP30). We compare the distribution of Mover to that of the synaptic vesicle protein synaptophysin, thereby determining the distribution of Mover in a quantitative manner relative to the abundance of synaptic vesicles. Notably, this method could potentially be implemented to allow for comparison of the distribution of proteins using different antibodies or microscopes or across different studies. Our method circumvents the inherent variability of immunofluorescent stainings by yielding a ratio rather than absolute fluorescence levels. Additionally, the method we describe enables the researcher to analyze the distribution of a protein on different levels: from whole brain slices to brain regions to different subregions in one brain area, such as the different layers of the hippocampus or sensory cortices. Mover is a vertebrate-specific protein that is associated with synaptic vesicles. With this method, we show that Mover is heterogeneously distributed across brain areas, with high levels in the ventral pallidum, the septal nuclei, and the amygdala, and also within single brain areas, such as the different layers of the hippocampus.

Here, we describe a quantitative approach to determining the distribution of a synaptic protein relative to a marker protein using immunofluorescence staining, confocal microscopy, and computer-based analysis.

The presence, absence, or levels of specific synaptic proteins can severely influence synaptic transmission. In addition to elucidating the function of a ...

Purification of Prominin-1+ Stem Cells from Postnatal Mouse Cerebellum

Most cerebellar neurons arise from two embryonic stem niches: a rhombic lip niche, which generates all the cerebellar excitatory glutamatergic neurons, and a ventricular zone niche, which generates the inhibitory GABAergic Purkinje cells, which are neurons that constitute the deep cerebellar nuclei and Bergman glia. Recently, a third stem cell niche has been described that arises as a secondary germinal zone from the ventricular zone niche. The cells of this niche are defined by the cell surface marker prominin-1 and are localized to the developing white matter of the postnatal cerebellum. This niche accounts for the late born molecular layer GABAergic interneurons along with postnatally generated cerebellar astrocytes. In addition to their developmental role, this niche is gaining translational importance in regards to its involvement in neurodegeneration and tumorigenesis. The biology of these cells has been difficult to decipher because of a lack of efficient techniques for their purification. Demonstrated here are efficient methods to purify, culture, and differentiate these postnatal cerebellar stem cells.

Purification of Prominin-1+ Stem Cells from Postnatal Mouse Cerebellum

Demonstrated here is an efficient and cost-effective method to purify, culture, and differentiate white matter stem cells from postnatal mouse cerebellum.

Most cerebellar neurons arise from two embryonic stem niches: a rhombic lip niche, which generates all the cerebellar excitatory glutamatergic neurons, ...