This work was supported by Ministerio de Econom&#236;a y Competitividad/Instituto de Salud Carlos III (SAF2014-55700-P, PCIN-2013-019-C03-03 and PIE14/00034), Instituci&#242; Catalana de Recerca i Estudis Avan&#231;ats (ICREA Academia-2010), and Agentschap voor Innovatie door Wetenschap en Technologie (SBO-140028) to F. C. Also, X. M, V. F.-D., and F. C. belong to the "Neuropharmacology and Pain" accredited research group (Generalitat de Catalunya,2014 SGR 1251). The work was also supported by the "Programa Pesquisador Visitante Especial-Ci&#234;ncia sem Fronteiras" from CAPES (Brazil) to F.C.

All animal experimental procedures were approved by the University of Barcelona Committee on Animal Use and Care (CEEA), in compliance with the guidelines described in the Guide for the Care and Use of Laboratory Animals11 and following the European Community, law 86/609/CCE, FELASA, and ARRIVE guidelines. Thus, mice are housed in standard cages, with ad libitum access to food and water, and are maintained under controlled standard conditions (12 h dark/light cycle starting at 7:30 AM, 22 &#176;C temperature, and 66% humidity).
1. Obtaining Mouse Brain Synaptosomes Using a Discontinuous Sucrose Gradient
Note: This method was reported previously10.
<ol>
	<li>Prepare fresh isolation buffer (IB), pH 7.4; 2 M sucrose; 1 M sucrose/0.1 mM CaCl2; and 0.1 mM CaCl2 (see Table 1). Chill the solutions on ice.</li>
	<li>Sacrifice five mice by cervical dislocation. Decapitate and rapidly remove the brain of each animal. Dissect the brain region of interest (i.e., the hippocampus, 0.25-0.35 g of fresh tissue) and place it in a 5 mL Potter-Elvehjem glass tube on ice with 1 mL of IB.</li>
	<li>Homogenize the tissue using a homogenizing stirrer (10 strokes at 700-900 rotations per min), preferentially in an ice bath to preclude sample warming.</li>
	<li>Place the homogenized tissue (1 mL) into a 15 mL tube containing 6 mL of 2 M sucrose and 2.5 mL of 0.1 mM CaCl2 at 4 &#176;C. Mix slowly by inverting the tube. 
	Note: The addition of calcium is essential to maintaining the adhesive interactions between organelles and thus to preserving the structure of the different &#34;densities.&#34;</li>
	<li>Place the solution in a 12 mL centrifuge tube and slowly add 2.5 mL of 1 M sucrose/0.1 mM CaCl2 on top of each tube to form the sucrose gradient. 
	Note: Label the position of the gradient interface on the centrifuge tube using a permanent marker.</li>
	<li>Weigh and equilibrate the centrifuge tubes with IB solution within their corresponding steel supports and with their closing lids.</li>
	<li>Centrifuge the tubes for 3 h at 100,000 x g and 4 &#176;C using a swinging bucket rotor centrifuge. Completely fill the rotor with all the steel supports (even empty, if required).</li>
	<li>Discard the top layer containing myelin. With a Pasteur pipette, collect the white ring between the 1.25-M and 1-M sucrose interphase corresponding to the synaptosome fraction.</li>
	<li>Dilute the synaptosomes with 9X their volume using IB in a centrifuge tube.</li>
	<li>Weigh and equilibrate the centrifuge tubes with IB solution within their corresponding steel supports and with their closing lids.</li>
	<li>Centrifuge the tubes for 30 min at 15,000 x g and 4 &#176;C using a swinging bucket rotor centrifuge.</li>
	<li>Discard the supernatant and resuspend the pellet with 1.1 mL of IB solution. Collect 100 &#181;L of this synaptosomal solution and centrifuge for 5 min at 11,000 x g and 4 &#186;C.</li>
	<li>Resuspend the synaptosomal pellet in 5% sodium dodecyl sulfate (SDS) and store this sample at -20 &#186;C. 
	Note: This sample will correspond to the total synaptosome fraction for further Western-blot analysis. The remaining synaptosomal fraction (~1 mL) can either be frozen down at -20 &#186;C until use or processed for subsequent subsynaptic fractionation. The synaptosomes or purified synapses represent between 1 and 2% of the total hippocampal volume12.</li>
</ol>
2. Pre-, Post- and Extrasynaptic-isolation
<ol>
	<li>Prepare 2x fresh solubilization buffer, pH 6.0; 1x solubilization buffer, pH 6.0; solubilization buffer, pH 8.0; and 0.1 mM CaCl2 (see Table 1). Chill the solutions on ice. 
	Note: The pH of these solutions should be accurately adjusted to achieve a good subsynaptic fractionation.</li>
	<li>Slowly dilute the 1-mL resuspended synaptosomal fraction (see step 1.12) with 5 mL of 0.1 mM CaCl2.</li>
	<li>Add the same volume (5 mL) of ice-cold 2x solubilization buffer, pH 6.0, and incubate for 50 min on ice under high agitation in a beaker on ice. 
	Note: While 1% of Triton X-100 at pH 6.0 solubilizes all plasma membrane proteins, it preserves proteins within the synaptic contacts or junctions (i.e., pre- and postsynaptic structures)4.</li>
	<li>Place the solution in a centrifuge tube. Weigh and equilibrate the tubes with equivalent volumes of 0.1 mM CaCl2 and 2x solubilization buffer solution, pH 6.0, within their corresponding steel supports and with their closing lids.</li>
	<li>Centrifuge the tubes for 30 min at 40,000 x g and 4 &#176;C using a swinging bucket rotor centrifuge. The resulting supernatant represents the extrasynaptic fraction, and the pellet corresponds to the synaptic junctions (i.e., pre- and postsynaptic fractions).</li>
	<li>Concentrate the supernatant containing the extrasynaptic fraction to a final 200 &#181;L volume using a 15 mL 10K filter tube centrifuged at 4,000 x g and 4 &#186;C.</li>
	<li>Precipitate the resulting concentrated extrasynaptic fraction (200 &#181;L) with 5 volumes (1 mL) of pre-chilled (-20 &#176;C) acetone overnight at -20 &#176;C.</li>
	<li>The next day, centrifuge the extrasynaptic fraction for 30 min at 18,000 x g and -15 &#176;C. After centrifugation, discard the acetone supernatant and dry the pellet to remove any trace of acetone.</li>
	<li>Finally, resuspend the pellet containing the extrasynaptic proteins with 200 &#181;L of 5% SDS. Sonicate the pellet, if required.</li>
	<li>Without disrupting it, carefully wash the pellet containing the pre- and postsynaptic fractions with 2 mL of 1x solubilization buffer, pH 6.0. Discard the buffer.</li>
	<li>Resuspend the pellet with 10 mL of 1x solubilization buffer, pH 8.0, using a glass Pasteur pipette. 
	Note: While 1% of Triton X-100 at pH 8.0 solubilizes the presynaptic specialization, it preserves the insoluble postsynaptic density4.</li>
	<li>Incubate the suspension for 50 min on ice under high agitation in a beaker on ice.</li>
	<li>Place the solution in a centrifuge tube. Weigh and equilibrate the tubes with 1x solubilization buffer solution, pH 8.0, within their corresponding steel supports and with their closing lids.</li>
	<li>Centrifuge the tubes for 30 min at 40,000 x g and 4 &#176;C using a swinging bucket rotor centrifuge; the resulting supernatant corresponds to the presynaptic fraction and the pellet to the postsynaptic fraction.</li>
	<li>Process the supernatant containing the presynaptic fraction, as described in steps 2.6-2.9.</li>
	<li>Resuspend the pellet containing postsynaptic fraction with 200 &#181;L of 5% SDS. Sonicate the pellet, if required.</li>
</ol>
3. Analyze the samples by immunoblot to validate the membrane fractionation
<ol>
	<li>Determine the amount of protein in each fraction (i.e., the total, extra-, pre- and post-synaptic fractions) using the bicinchoninic acid protein assay.</li>
	<li>Take 20 &#181;g of protein from each fraction and dilute it to a final volume of 50 &#181;L in SDS-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. Boil for 5 min at 100 &#186;C.</li>
	<li>Separate the proteins by SDS-PAGE electrophoresis 10%, with a 4% concentrating gel under reducing conditions. Electrophorese at constant voltage of 80 V until the dye enters the lower gel and then increase the voltage to 120 V.</li>
	<li>Transfer the proteins to a PVDF membrane and block with IB blocking solution for 45 min at room temperature under continuous shaking.</li>
	<li>Incubate the membrane overnight at 4 &#186;C with the indicated primary antibody (i.e., anti-SNAP-25, anti-PSD-95, anti-synaptophysin, or anti-GPR37) diluted in IB blocking solution under continuous shaking.</li>
	<li>Wash the membrane three times (10 min each) with IB washing solution to eliminate unbound primary antibody.</li>
	<li>Incubate with the indicated secondary antibody for 90 min in dark conditions at room temperature under continuous shaking.</li>
	<li>Wash the membrane three times (10 min each) with IB washing solution to eliminate unbound secondary antibody.</li>
	<li>Incubate the membrane with chemiluminescent substrate (prepare the mix under dark conditions and following the 1:1 proportion of solution A and B provided by the manufacturer).</li>
	<li>Analyze the membrane in a chemiluminescent detection device.</li>
</ol>

<ol>
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R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+presynaptic+particle+web:+ultrastructure,+composition,+dissolution,+and+reconstitution.">The presynaptic particle web: ultrastructure, composition, dissolution, and reconstitution.</a> Neuron. 32, 63-77 (2001).</li><li>Pinheiro, P. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solubilization+and+immunological+identification+of+presynaptic+alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic+acid+receptors+in+the+rat+hippocampus.">Solubilization and immunological identification of presynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors in the rat hippocampus.</a> Neurosci. Lett. 336, 97-100 (2003).</li><li>Rebola, N., Pinheiro, P. C., Oliveira, C. R., Malva, J. O., Cunha, R. 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The authors have nothing to disclose.

The protocol presented here constitutes a powerful biochemical tool for the study of the subsynaptic distribution of specific proteins within any brain region. However, there are some drawbacks inherent to the technique that deserve to be highlighted here. For instance, one of the main limitations is the relatively large amount of tissue needed to purify a reasonable amount of protein in order to perform the immunoblot analysis of all subsynaptic fractions. This issue might be related to the fact that synapses (i.e., synaptosomes) represent only 1-2% of the total hippocampal volume12. Indeed, between 1 and 1.5 g of fresh tissue (i.e., the hippocampus) is needed to perform a successful fractionation; otherwise, the yield is too low to assess the identity, as well as the localization, of the protein being studied. 
Conversely, if an excess of brain tissue is used, then the separation procedure will not be optimal. It is also critical to carefully adjust the pH of the solutions to ensure an optimal subsynaptic fractionation. Consequently, every time a brain membrane fractionation is performed, it is mandatory to validate the efficiency of each fraction before any further study. Importantly, the following protocol it is unlikely to be suitable for the subsynaptic fractionation of inhibitory synapses because of its differential structure and distribution. However, all these drawbacks do not overshadow the enormous usefulness of this experimental procedure, which, without any doubt, will become a universal methodology for the study of the synapse.

Synaptic transmission relies on the physical integrity of the synapse, a concept that was envisaged as early as 1897 by Foster and Sherrington1. Thus, understanding the distribution of key neurotransmission components (e.g., ion channels, receptors, etc.) is essential to elucidate synaptic function, both in normal and pathological conditions. Electron microscopy (EM) has contributed enormously to the current ultrastructural notion of prototypical central nervous system (CNS) synapses. In such a way, EM has finely established the differences between pre- and postsynaptic densities, which are separated by a cleft of a rather uniform distance (~25 nm)2. Interestingly, the postsynaptic apparatus exhibits a relatively continuous, electron-dense thickening below its plasma membrane, the so-called postsynaptic density, or PSD2. Conversely, at the presynaptic apparatus, a noticeable discontinuous cytomatrix network is arranged just beneath the plasma membrane, which is essential to the alignment and docking of synaptic vesicles to the plasma membrane active zone3. Hence, EM constitutes the golden experimental approach to survey the distribution of proteins within structurally preserved CNS synapses. However, the information provided by electron micrographs is static. Indeed, accumulating evidences show that in vivo synapses are extremely dynamic, thus experiencing dramatic structural changes upon sustained synaptic transmission. In addition, the morphology and composition of synapses can change throughout different CNS regions and upon development, maturation, aging, and the development of neuropathological conditions. Overall, a protocol focused on isolating proteins belonging to different synaptic compartments in physiological conditions represents a valuable tool for a more comprehensive study of synaptic functioning.
Here, we describe this kind of complementary experimental approach, which allows for the preparative biochemical enrichment of the different synaptic membrane compartments-namely, extra-, pre- and postsynaptic membrane domains. This membrane fractionation method, first described by Philips et al. (2001)4, is based on a pH shift that weakens the adhesive interactions occurring within the pre- and postsynaptic apparatus. First, by using mild detergents at pH 6.0, it is possible to discern the adherens junction that holds the pre- and postsynaptic apparatus and that is maintained from the extrasynaptic membrane domain, which is solubilized and thus can be extracted from the synaptic contacts. Subsequently, raising the pH from 6.0 to 8.0 in the presence of mild detergents weakens the strength of the adherens junction that keeps the presynaptic active zone tightly bound to the postsynaptic density. Hence, the presynaptic compartment is solubilized and can be separated from the postsynaptic density, which is mostly preserved because the concentration of detergent used does not promote its solubilization4. Interestingly, the fractionation efficiency, eventually higher than 90%, can be confirmed by different subsynaptic markers: i) synaptosomal-associated protein 25 (SNAP-25), from the presynaptic active zone; ii) synaptophysin, from the extrasynaptic fraction (i.e., outside the active zone and including microsomes); and iii) postsynaptic density protein 95 (PSD-95), from the postsynaptic density. Notably, this brain membrane fractionation method has been used successfully. Accordingly, it has been possible to precisely determine the subsynaptic localization of different receptors, such as alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors5, adenosine A1 receptor (A1R)6, adenosine A2A receptor (A2AR)7, adenosine triphosphate (ATP) P2 receptors8, nicotinic acetylcholine receptor subunits9, and Parkinson&#39;s disease-associated receptor GPR3710. However, a number of limitations may impede the proper assessment of the synaptic distribution of a particular neuronal protein. Thus, in this procedure, we not only fully describe the entire protocol, but we also highlight some critical points to be considered, such as the rather large amount of tissue needed, the low protein yield, and the mandatory requirement to validate the efficiency of each separation before performing the definite experiment.

<table border="0" cellpadding="0" cellspacing="0" width="704">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Sucrose</td>
			<td style="width:249px;">Pancreac Qu&#237;mica SL, Barcelona, Spain</td>
			<td style="width:160px;">1,316,211,211</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">CaCl2</td>
			<td style="width:249px;">Pancreac Qu&#237;mica SL, Barcelona, Spain</td>
			<td style="width:160px;">2,112,211,210</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">MgCl2&#183;6H2O</td>
			<td style="width:249px;">Pancreac Qu&#237;mica SL, Barcelona, Spain</td>
			<td style="width:160px;">1,313,961,210</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:128px;">Protease inhibitor cocktail Set III</td>
			<td style="width:249px;">Millipore, Darmstadt, Germany</td>
			<td style="width:160px;">535140</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Trizma Base</td>
			<td style="width:249px;">Sigma, St. Louis, MO, USA</td>
			<td style="width:160px;">T1503</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Tris-HCl</td>
			<td style="width:249px;">Pancreac Qu&#237;mica SL, Barcelona, Spain</td>
			<td style="width:160px;">1,236,541,209</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Triton X-100</td>
			<td style="width:249px;">Sigma, St. Louis, MO, USA</td>
			<td style="width:160px;">X100</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">SDS</td>
			<td style="width:249px;">Sigma, St. Louis, MO, USA</td>
			<td style="width:160px;">L3771</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Glycerol</td>
			<td style="width:249px;">Sigma, St. Louis, MO, USA</td>
			<td style="width:160px;">G5516</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Bromophenol Blue</td>
			<td style="width:249px;">Pancreac Qu&#237;mica SL, Barcelona, Spain</td>
			<td style="width:160px;">1,311,651,604</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Dithiothreitol</td>
			<td style="width:249px;">Sigma, St. Louis, MO, USA</td>
			<td style="width:160px;">D0632</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Tween 20</td>
			<td style="width:249px;">Sigma, St. Louis, MO, USA</td>
			<td style="width:160px;">P2287</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Non fat dry milk</td>
			<td style="width:249px;"></td>
			<td style="width:160px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">NaCl</td>
			<td style="width:249px;">Pancreac Qu&#237;mica SL, Barcelona, Spain</td>
			<td style="width:160px;">1,216,591,211</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">KCl</td>
			<td style="width:249px;">Pancreac Qu&#237;mica SL, Barcelona, Spain</td>
			<td style="width:160px;">1,314,941,210</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">KH2PO4</td>
			<td style="width:249px;">Merck</td>
			<td style="width:160px;">4873</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Na2HPO4</td>
			<td style="width:249px;">Pancreac Qu&#237;mica SL, Barcelona, Spain</td>
			<td style="width:160px;">1,316,781,211</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Basic 20 pH</td>
			<td style="width:249px;">Crison, Alella, Spain</td>
			<td style="width:160px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:128px;">Polytron VDI 12 Adaptable Homogenizer</td>
			<td style="width:249px;">VWR, Radnor, PA, USA.</td>
			<td style="width:160px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="65">
			<td height="65" style="height:65px;width:128px;">Ultra-Clear Tubes (14x89mm)</td>
			<td style="width:249px;">Beckman Coulter, Hospitalet de Llobregat, Barcelona</td>
			<td style="width:160px;">344059</td>
			<td style="width:167px;">Tubes should be filled almost completely when used to prevent collapsing due to ultracentrifugation.</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:128px;">Amicon Ultra-15 Centrifugal filters Ultracel -10K</td>
			<td style="width:249px;">Merck Millipore, Darmstadt, Germany</td>
			<td style="width:160px;">UFC901008</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Centrifuge 5430R</td>
			<td style="width:249px;">Eppendorf, Hamburgo, Germany</td>
			<td style="width:160px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:128px;">Optima L-90K Ultracentrifuge</td>
			<td style="width:249px;">Beckman Coulter, Hospitalet de Llobregat, Barcelona</td>
			<td style="width:160px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Sonifier 250</td>
			<td style="width:249px;">Branson, Danbury, Connecticut</td>
			<td style="width:160px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Amersham Imager 600</td>
			<td style="width:249px;">GE Healthcare Europe GmbH, Barcelona, Spain</td>
			<td style="width:160px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:128px;">Disposable Glass Pasteur Pippetes 230 mm</td>
			<td style="width:249px;">VWR, Radnor, PA, USA</td>
			<td style="width:160px;">612-1702</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:128px;">Compact Balance EK-610</td>
			<td style="width:249px;">A&#38;D, Tokyo, Japan</td>
			<td style="width:160px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:128px;">Pierce&#8482; BCA Protein Assay Kit</td>
			<td style="width:249px;">Pierce Biotechnology, Rockford, IL, USA</td>
			<td style="width:160px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:128px;">SuperSignal west pico chemiluminescent substrate</td>
			<td style="width:249px;">Thermo Fisher Scientific Inc., Waltham, MA, USA</td>
			<td style="width:160px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:128px;">GR 200 Precision Balance</td>
			<td style="width:249px;">A&#38;D, Tokyo, Japan</td>
			<td style="width:160px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:128px;">Anti-GPR37</td>
			<td style="width:249px;">Homemade antibody anti-GPR37 produced and validated in Francisco Ciruela Laboratory.</td>
			<td style="width:160px;"></td>
			<td style="width:167px;">Primary antibodies used at a final concentration of 0.250ug/ml</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:128px;">Anti-SNAP-25, anti-PSD-95, anti-synaptophysin</td>
			<td style="width:249px;">Abcam, Cambridge, United Kingdom</td>
			<td style="width:160px;"></td>
			<td style="width:167px;">Primary antibodies diluted 1:10000</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:128px;">HRP-conjugated goat anti-mouse IgG</td>
			<td style="width:249px;">Thermo Fisher Scientific, Waltham, MA, USA.</td>
			<td style="width:160px;"></td>
			<td style="width:167px;">Secondary antibody diluted 1:10000</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:128px;">HRP-conjugated goat anti-rabbit IgG</td>
			<td style="width:249px;">Thermo Fisher Scientific, Waltham, MA, USA.</td>
			<td style="width:160px;"></td>
			<td style="width:167px;">Secondary antibody diluted 1:30000</td>
		</tr>
	</tbody>
</table>

brain membrane fractionation: an ex vivo approach to assess subsynaptic protein localization

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate neurotransmission that occurs within synapses because it is highly regulated by dynamic protein-protein interactions and phosphorylation events. Accordingly, when any method is used to study synaptic transmission, a major goal is to preserve these transient physiological modifications. Here, we present a brain membrane fractionation protocol that represents a robust procedure to isolate proteins belonging to different synaptic compartments. In other words, the protocol describes a biochemical methodology to carry out protein enrichment from presynaptic, postsynaptic, and extrasynaptic compartments. First, synaptosomes, or synaptic terminals, are obtained from neurons that contain all synaptic compartments by means of a discontinuous sucrose gradient. Of note, the quality of this initial synaptic membrane preparation is critical. Subsequently, the isolation of the different subsynaptic compartments is achieved with light solubilization using mild detergents at differential pH conditions. This allows for separation by gradient and isopycnic centrifugations. Finally, protein enrichment at the different subsynaptic compartments (i.e., pre-, post- and extrasynaptic membrane fractions) is validated by means of immunoblot analysis using well-characterized synaptic protein markers (i.e., SNAP-25, PSD-95, and synaptophysin, respectively), thus enabling a direct assessment of the synaptic distribution of any particular neuronal protein.

The described methodology has been largely used for the subsynaptic analysis of neuronal proteins in general and for the isolation and biochemical characterization of synaptic receptors5,6,7,8,9 in particular. Interestingly, the representative result displayed here show the usefulness of this experimental procedure for the analysis of the subsynaptic hippocampal distribution of an orphan G protein-coupled receptor- namely, the Parkinson&#39;s disease-associated receptor GPR3710. GPR37 was originally identified through searches for homologues for endothelin and bombesin receptors13, although it was found not to bind to endothelins or related peptides. In its place, GPR37 was proposed to be activated by the head activator peptide14,15,16 and, more recently, by the neuropeptides prosaposin and prosaptide17, although these associations are yet to be universally accepted. GPR37 has received the most attention for its linkage to Parkinson&#39;s disease18. Thus, there is significant interest in knowing the neurobiology of this intriguing receptor, both under normal and pathological conditions. Therefore, uncovering the GPR37 subsynaptic localization might help to clarify its function within the brain. To this end, the hippocampus was first isolated from C57BL/6J (WT) and GPR37-KO mice at eight weeks of age. Then, the extra-, pre- and post-subsynaptic fractions were purified using the membrane fractionation protocol (see Figure 1 for a schematic overview of the procedure). Subsequently, the purities of these subsynaptic compartments were verified by the segregation of the respective synaptic markers: i) the extrasynaptic vesicular marker (synaptophysin); ii) the presynaptic active zone marker (SNAP-25); and iii) the postsynaptic density marker (PSD95). Accordingly, the enrichments in synaptophysin, SNAP-25, and PSD95 within the extra-, pre- and post-subsynaptic fractions, respectively, were analyzed by immunoblot using specific antibodies against these proteins (Figure 2). Hence, a fractionation efficiency of at least 90% was found for each synaptic marker tested (Figure 3), similar to those described previously6. Interestingly, GPR37 immunoreactivity was more abundant (n = 3; P &#60;0.001) in the extrasynaptic fraction when compared to the presynaptic (20 &#177; 4%) and the postsynaptic (36 &#177; 2%) fractions (Figure 3). In addition, our data indicated that, while present at the presynaptic active zone, GPR37 was mainly localized in the postsynaptic density (n = 3; P &#60;0.05) (Figure 3). Overall, the brain membrane fractionation protocol allowed for the assessment of the subsynaptic distribution of GPR37 in the mouse hippocampus, thus providing valuable information for future manipulations of this orphan receptor.
<img alt="Figure 1" src="/files/ftp_upload/55661/55661fig1.jpg" /> 
Figure 1: Schematic flow-chart representation of the membrane fractionation protocol. All experimental procedures are described in the left-hand column, while the sample collection is depicted in the right-hand column. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55661/55661fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55661/55661fig2.jpg" /> 
Figure 2: Subsynaptic distribution of GPR37 in the mouse hippocampus. Representative immunoblot showing Synaptophysin, SNAP-25 and PSD-95 as extra-, pre- and postsynaptic specific synaptic markers, as well as GPR37 immunoreactivity in hippocampal synaptic fractions of WT and GPR37-KO mice. Hippocampal synaptosomes (Syn) were fractionated into extrasynaptic (Extra) and presynaptic (Pre) active zones and postsynaptic density (Post) fractions. These were analyzed by immunoblot (20 &#181;g of protein/lane) using the rabbit anti-synaptophysin (1:3,000), mouse anti-SNAP-25 (1:3,000), rabbit anti-PSD95 (1:3,000), and rabbit anti-GPR37 (1 &#181;g/mL) antibodies. The primary bound antibody was detected using either an HRP-conjugated goat anti-rabbit (1/30,000) or a rabbit anti-mouse (1:30,000) antibody. These data are extracted from reference10, with permission. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55661/55661fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55661/55661fig3.jpg" /> 
Figure 3: Relative quantification of GPR37 enrichment in hippocampal extra-, pre-, and postsynaptic fractions. The intensities of the immunoreactive bands on the immunoblotted membranes corresponding to extrasynaptic (Extra; yellow column), presynaptic (Pre, green column), and postsynaptic (Post, red column) fractions, shown in Figure 2, were measured by densitometric scanning. The densities were quantified from non-saturated bands. Values were normalized (in % of the relative densitometric scanning, RDS) using the amount of Synaptophysin, SNAP-25, PSD95, and GPR37 in the most enriched fraction and were presented as the means &#177; SEM of three independent experiments10. The asterisks denote significantly different data: *p &#60;0.05, ***p &#60;0.001 (1-way ANOVA with a Bonferroni&#39;s post hoc test). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55661/55661fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization at JoVE.com

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Here, we present a brain membrane fractionation protocol that represents a robust procedure to isolate proteins belonging to different synaptic compartments.

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate ...

brain-membrane-fractionation-an-ex-vivo-approach-to-assess

Nanyang Technological University

Research

JoVE Journal

Biochemistry

10.3K Views.  Universitat de Barcelona, L'Hospitalet de Llobregat. Here, we present a brain membrane fractionation protocol that represents a robust procedure to isolate proteins belonging to different synaptic compartments.

Video: Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and  GTPase-linked Immunosorbent Assay

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of diverse cellular functions, including cytoskeletal dynamics, cell motility, cell polarity, axonal guidance, vesicular trafficking, and cell cycle control. Changes in Rho GTPase signaling play an essential regulatory role in many pathological conditions, such as&#160;cancer, central nervous system diseases, and immune system-dependent diseases. The posttranslational modification of Rho GTPases (i.e., prenylation by mevalonate pathway intermediates) and GTP binding are key factors which affect the activation of this protein. In this paper, two essential and simple methods are provided to detect a broad range of Rho GTPase prenylation and GTP binding activities. Details of the technical procedures that have been used are explained step by step in this manuscript.

Here we describe a protocol to investigate the prenylation and guanosine-5'-triphosphate (GTP)-loading of Rho GTPase. This protocol consists of two detailed methods, namely membrane fractionation and a GTPase-linked immunosorbent assay. The protocol can be used for measuring the prenylation and GTP loading of different other small GTPases.

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of ...

Analysis of Thylakoid Membrane Protein Complexes by Blue Native Gel Electrophoresis

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded in the thylakoid membrane harvest solar energy to drive electrons from water to NADP+ via two photosystems, and use the created proton gradient for production of ATP. Photosystem PSII, PSI, cytochrome b6f (Cyt b6f) and ATPase are all multiprotein complexes with distinct orientation and dynamics in the thylakoid membrane. Valuable information about the composition and interactions of the protein complexes in the thylakoid membrane can be obtained by solubilizing the complexes from the membrane integrity by mild detergents followed by native gel electrophoretic separation of the complexes. Blue native polyacrylamide gel electrophoresis (BN-PAGE) is an analytical method used for the separation of protein complexes in their native and functional form. The method can be used for protein complex purification for more detailed structural analysis, but it also provides a tool to dissect the dynamic interactions between the protein complexes. The method was developed for the analysis of mitochondrial respiratory protein complexes, but has since been optimized and improved for the dissection of the thylakoid protein complexes. Here, we provide a detailed up-to-date protocol for analysis of labile photosynthetic protein complexes and their interactions in Arabidopsis thaliana.

A protocol for the elucidation of plant thylakoid protein complex organization and composition with blue native polyacrylamide gel electrophoresis (BN-PAGE) and 2D-SDS-PAGE is described. The protocol is optimized for Arabidopsis thaliana, but can be used for other plant species with minor modifications.

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded ...

Preparation of Chloroplast Sub-compartments from Arabidopsis for the Analysis of Protein Localization by Immunoblotting or Proteomics

Chloroplasts are major components of plant cells. Such plastids fulfill many crucial functions, such as assimilation of carbon, sulfur and nitrogen as well as synthesis of essential metabolites. These organelles consist of the following three key sub-compartments. The envelope, characterized by two membranes, surrounds the organelle and controls the communication of the plastid with other cell compartments. The stroma is the soluble phase of the chloroplast and the main site where carbon dioxide is converted into carbohydrates. The thylakoid membrane is the internal membrane network consisting of grana (flat compressed sacs) and lamellae (less dense structures), where oxygenic photosynthesis takes place. The present protocol describes step by step procedures required for the purification, using differential centrifugations and Percoll gradients, of intact chloroplasts from Arabidopsis, and their fractionation, using sucrose gradients, in three sub-compartments (i.e., envelope, stroma, and thylakoids). This protocol also provides instructions on how to assess the purity of these fractions using markers associated to the various chloroplast sub-compartments. The method described here is valuable for subplastidial localization of proteins using immunoblotting, but also for subcellular and subplastidial proteomics and other studies.

Here, we describe a method to purify intact chloroplasts from Arabidopsis leaves and their three main sub-compartments (envelope, stroma, and thylakoids), using a combination of differential centrifugations, continuous Percoll gradients, and discontinuous sucrose gradients. The method is valuable for subplastidial and subcellular localization of proteins by immunoblotting and proteomics.

Chloroplasts are major components of plant cells. Such plastids fulfill many crucial functions, such as assimilation of carbon, sulfur and nitrogen as ...

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

Evaluation of Protein&#8211;Protein Interactions using an On-Membrane Digestion Technique

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions largely depend on intracellular protein&#8211;protein interactions. Therefore, research regarding these interactions is indispensable to facilitating the understanding of physiologic processes. Co-precipitation of associated proteins, followed by mass spectrometry (MS) analysis, enables the identification of novel protein interactions. In this study, we have provided details of the novel technique of immunoprecipitation-liquid chromatography (LC)-MS/MS analysis combined with on-membrane digestion for the analysis of protein&#8211;protein interactions. This technique is suitable for crude immunoprecipitants and can improve the throughput of proteomic analyses. Tagged recombinant proteins were precipitated using specific antibodies; next, immunoprecipitants blotted onto polyvinylidene difluoride membrane pieces were subjected to reductive alkylation. Following trypsinization, the digested protein residues were analyzed using LC-MS/MS. Using this technique, we were able to identify several candidate associated proteins. Thus, this method is convenient and useful for the characterization of novel protein&#8211;protein interactions.

Here, we present a protocol for an on-membrane digestion technique for the preparation of samples for mass spectrometry. This technique facilitates the convenient analysis of protein&#8211;protein interactions.

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions ...

Measuring Interactions between Fluorescent Probes and Lignin in Plant Sections by sFLIM Based on Native Autofluorescence

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis process, which maintains enzymes far from their substrate. Characterization of these complex interactions is thus a challenge in complex substrates such as LB. The method here measures molecular interactions between fluorophore-tagged molecules and native autofluorescent lignin, to be revealed by F&#246;rster resonance energy transfer (FRET). Contrary to FRET measurements in living cells using two exogenous fluorophores, FRET measurements in plants using lignin is not trivial due to its complex autofluorescence. We have developed an original acquisition and analysis pipeline with correlated observation of two complementary properties of fluorescence: fluorescence emission and lifetime. sFLIM (spectral and fluorescent lifetime imaging microscopy) provides the quantification of these interactions with high sensitivity, revealing different interaction levels between biomolecules and lignin.

This protocol describes an original setup combining spectral and fluorescence lifetime measurements to evaluate F&#246;rster resonance energy transfer (FRET) between rhodamine-based fluorescent probes and lignin polymer directly in thick plant sections.

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis ...

Native Cell Membrane Nanoparticles System for Membrane Protein-Protein Interaction Analysis

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal transduction; from sensing environmental signals to biological response; from metabolic regulation to developmental control. Accurate structural information of protein-protein interactions is crucial for understanding the molecular mechanisms of membrane protein complexes and for the design of highly specific molecules to modulate these proteins. Many in vivo and in vitro approaches have been developed for the detection and analysis of protein-protein interactions. Among them the structural biology approach is unique in that it can provide direct structural information of protein-protein interactions at the atomic level. However, current membrane protein structural biology is still largely limited to detergent-based methods. The major drawback of detergent-based methods is that they often dissociate or denature membrane protein complexes once their native lipid bilayer environment is removed by detergent molecules. We have been developing a&#160;native cell membrane nanoparticle system for membrane protein structural biology. Here, we demonstrate the use of this system in the analysis of protein-protein interactions on the cell membrane with a case study of the oligomeric state of AcrB.

Presented here is a protocol for the determination of oligomeric state of membrane proteins that utilizes a native cell membrane nanoparticle system in conjunction with electron microscopy.

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal ...

Polysome Profiling without Gradient Makers or Fractionation Systems

Polysome fractionation by sucrose density gradient centrifugation is a powerful tool that can be used to create ribosome profiles, identify specific mRNAs being translated by ribosomes, and analyze polysome associated factors. While automated gradient makers and gradient fractionation systems are commonly used with this technique, these systems are generally expensive and can be cost-prohibitive for laboratories that have limited resources or cannot justify the expense due to their infrequent or occasional need to perform this method for their research. Here, a protocol is presented to reproducibly generate polysome profiles using standard equipment available in most molecular biology laboratories without specialized fractionation instruments. Moreover, a comparison of polysome profiles generated with and without a gradient fractionation system is provided. Strategies to optimize and produce reproducible polysome profiles are discussed. Saccharomyces cerevisiae is utilized as a model organism in this protocol. However, this protocol can be easily modified and adapted to generate ribosome profiles for many different organisms and cell types.

This protocol describes how to generate a polysome profile without using automated gradient makers or gradient fractionation systems.

Polysome fractionation by sucrose density gradient centrifugation is a powerful tool that can be used to create ribosome profiles, identify specific mRNAs ...

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Despite recent advances in the characterization of yeast mitochondrial proteome, the submitochondrial localization of a significant number of proteins remains elusive. Here, we describe a robust and effective method for determining the suborganellar localization of yeast mitochondrial proteins, which is considered a fundamental step during mitochondrial protein function elucidation. This method involves an initial step that consists of obtaining highly pure intact mitochondria. These mitochondrial preparations are then subjected to a subfractionation protocol consisting of hypotonic shock (swelling) and incubation with proteinase K (protease). During swelling, the outer mitochondrial membrane is selectively disrupted, allowing the proteinase K to digest proteins of the intermembrane space compartment. In parallel, to obtain information about the topology of membrane proteins, the mitochondrial preparations are initially sonicated, and then subjected to alkaline extraction with sodium carbonate. Finally, after centrifugation, the pellet and supernatant fractions from these different treatments are analyzed by SDS-PAGE and western blot. The submitochondrial localization as well as the membrane topology of the protein of interest is obtained by comparing its western blot profile with known standards.

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Despite recent advances, many yeast mitochondrial proteins still remain with their functions completely unknown. This protocol provides a simple and reliable method to determine the submitochondrial localization of proteins, which has been fundamental for the elucidation of their molecular functions.

Despite recent advances in the characterization of yeast mitochondrial proteome, the submitochondrial localization of a significant number of proteins ...

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

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Chapters in this video

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and GTPase-linked Immunosorbent Assay

Analysis of Thylakoid Membrane Protein Complexes by Blue Native Gel Electrophoresis

Preparation of Chloroplast Sub-compartments from Arabidopsis for the Analysis of Protein Localization by Immunoblotting or Proteomics

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

Evaluation of Protein–Protein Interactions using an On-Membrane Digestion Technique

Measuring Interactions between Fluorescent Probes and Lignin in Plant Sections by sFLIM Based on Native Autofluorescence

Native Cell Membrane Nanoparticles System for Membrane Protein-Protein Interaction Analysis

Polysome Profiling without Gradient Makers or Fractionation Systems

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae