Name: preparation of synaptic plasma membrane and postsynaptic density proteins using a discontinuous sucrose gradient
Uploaded: 2014-09-03T15:00:00
Description: Watch this Scientific Journal Video about Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient at JoVE.com

The authors thank Dr. Mike Ehlers and his laboratory members for originally demonstrating the protocol for subcellular fractionation of synaptic plasma membranes. We also thank Wendy Horsfall for management of the mouse colonies. This work was supported by operating grants from CIHR (AJR and AS).

The following protocol conforms to the guidelines of the Canadian Council of Animal Care and has been approved by the Faculty of Medicine and Pharmacy Animal Care Committee at the University of Toronto.
1. Prepare Required Reagents as Described in Table 1
<ol>
	<li>Add protease and phosphatase (if necessary) inhibitors to all sucrose solutions, buffers and ddH2O at concentrations outlined in Table 1. IMPORTANT: Perform all of the steps at 4&#160;&...

<ol>
	<li>Delint-Ramirez, I., 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=In+vivo+composition+of+NMDA+receptor+signaling+complexes+differs+between+membrane+subdomains+and+is+modulated+by+PSD-95+and+PSD-93.">In vivo composition of NMDA receptor signaling complexes differs between membrane subdomains and is modulated by PSD-95 and PSD-93.</a> The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. 30, 8162-8170 (2010).</li><li>Wang, Q., 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+psychiatric+disease+risk+factors+DISC1+and+TNIK+interact+to+regulate+synapse+composition+and+function.">The psychiatric disease risk factors DISC1 and TNIK interact to regulate synapse composition and function.</a> Mol Psychiatry. 16, (2010).</li><li>Ehlers, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reinsertion+or+degradation+of+AMPA+receptors+determined+by+activity-dependent+endocytic+sorting.">Reinsertion or degradation of AMPA receptors determined by activity-dependent endocytic sorting.</a> Neuron. 28, 511-525 (2000).</li><li>Tomita, S., Fukata, M., Nicoll, R. A., Bredt, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+interaction+of+stargazin-like+TARPs+with+cycling+AMPA+receptors+at+synapses.">Dynamic interaction of stargazin-like TARPs with cycling AMPA receptors at synapses.</a> Science. 303, 1508-1511 (2004).</li><li>Ehlers, M. D., Heine, M., Groc, L., Lee, M. C., Choquet, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusional+trapping+of+GluR1+AMPA+receptors+by+input-specific+synaptic+activity.">Diffusional trapping of GluR1 AMPA receptors by input-specific synaptic activity.</a> Neuron. 54, 447-460 (2007).</li><li>Bats, C., Groc, L., Choquet, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interaction+between+Stargazin+and+PSD-95+regulates+AMPA+receptor+surface+trafficking.">The interaction between Stargazin and PSD-95 regulates AMPA receptor surface trafficking.</a> Neuron. 53, 719-734 (2007).</li><li>Ehlers, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity+level+controls+postsynaptic+composition+and+signaling+via+the+ubiquitin-proteasome+system.">Activity level controls postsynaptic composition and signaling via the ubiquitin-proteasome system.</a> Nat Neurosci. 6, 231-242 (2003).</li><li>Cartier, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+biochemical+and+functional+protein+complex+involving+dopamine+synthesis+and+transport+into+synaptic+vesicles.">A biochemical and functional protein complex involving dopamine synthesis and transport into synaptic vesicles.</a> J Biol Chem. 285, 1957-1966 (2010).</li><li>Rodriguez de Lores, A., Alberici, M., De Robertis, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructural+and+enzymic+studies+of+cholinergic+and+non-cholinergic+synaptic+membranes+isolated+from+brain+cortex.">Ultrastructural and enzymic studies of cholinergic and non-cholinergic synaptic membranes isolated from brain cortex.</a> Journal of Neurochemistry. 14, 215-225 (1967).</li><li>Gray, E. G., Whittaker, V. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+isolation+of+nerve+endings+from+brain:+an+electron-microscopic+study+of+cell+fragments+derived+by+homogenization+and+centrifugation.">The isolation of nerve endings from brain: an electron-microscopic study of cell fragments derived by homogenization and centrifugation.</a> J Anat. 96, 79-88 (1962).</li><li>Cotman, C. W., Taylor, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+structural+studies+on+synaptic+complexes+from+rat+brain.">Isolation and structural studies on synaptic complexes from rat brain.</a> J Cell Biol. 55, 696-711 (1972).</li><li>Cotman, C. W., Banker, G., Churchill, L., Taylor, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+postsynaptic+densities+from+rat+brain.">Isolation of postsynaptic densities from rat brain.</a> The Journal of Cell Biology. 63, 441-455 (1974).</li><li>Carlin, R. K., Grab, D. J., Cohen, R. S., Siekevitz, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+postsynaptic+densities+from+various+brain+regions:+enrichment+of+different+types+of+postsynaptic+densities.">Isolation and characterization of postsynaptic densities from various brain regions: enrichment of different types of postsynaptic densities.</a> The Journal of Cell Biology. 86, 831-845 (1980).</li><li>Jones, D. H., Matus, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+synaptic+plasma+membrane+from+brain+by+combined+flotation-sedimentation+density+gradient+centrifugation.">Isolation of synaptic plasma membrane from brain by combined flotation-sedimentation density gradient centrifugation.</a> Biochim Biophys Acta. 356, 276-287 (1974).</li><li>Kennedy, M. B., Bennett, M. K., Erondu, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+and+immunochemical+evidence+that+the+"major+postsynaptic+density+protein"+is+a+subunit+of+a+calmodulin-dependent+protein+kinase.">Biochemical and immunochemical evidence that the "major postsynaptic density protein" is a subunit of a calmodulin-dependent protein kinase.</a> Proceedings of the National Academy of Sciences of the United States of America. 80, 7357-7361 (1983).</li><li>Moon, I. S., Apperson, M. L., Kennedy, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+major+tyrosine-phosphorylated+protein+in+the+postsynaptic+density+fraction+is+N-methyl-D-aspartate+receptor+subunit+2B.">The major tyrosine-phosphorylated protein in the postsynaptic density fraction is N-methyl-D-aspartate receptor subunit 2B.</a> Proceedings of the National Academy of Sciences of the United States of America. 91, 3954-3958 (1994).</li><li>Cho, K. O., Hunt, C. A., Kennedy, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+rat+brain+postsynaptic+density+fraction+contains+a+homolog+of+the+Drosophila+discs-large+tumor+suppressor+protein.">The rat brain postsynaptic density fraction contains a homolog of the Drosophila discs-large tumor suppressor protein.</a> Neuron. 9, 929-942 (1992).</li><li>Salahpour, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+amphetamine-induced+hyperactivity+and+reward+in+mice+overexpressing+the+dopamine+transporter.">Increased amphetamine-induced hyperactivity and reward in mice overexpressing the dopamine transporter.</a> Proc Natl Acad Sci U S A. 105, 4405-4410 (2008).</li><li>Ma, D. K., 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=Neuronal+activity-induced+Gadd45b+promotes+epigenetic+DNA+demethylation+and+adult+neurogenesis.">Neuronal activity-induced Gadd45b promotes epigenetic DNA demethylation and adult neurogenesis.</a> Science. 323, 1074-1077 (2009).</li><li>Budreck, E. C., Scheiffele, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroligin-3+is+a+neuronal+adhesion+protein+at+GABAergic+and+glutamatergic+synapses.">Neuroligin-3 is a neuronal adhesion protein at GABAergic and glutamatergic synapses.</a> The European Journal of Neuroscience. 26, 1738-1748 (2007).</li><li>Wu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arc/Arg3.1+regulates+an+endosomal+pathway+essential+for+activity-dependent+beta-amyloid+generation.">Arc/Arg3.1 regulates an endosomal pathway essential for activity-dependent beta-amyloid generation.</a> Cell. 147, 615-628 (2011).</li><li>Huttner, W. B., Schiebler, W., Greengard, P., De Camilli, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synapsin+I+(protein+I),+a+nerve+terminal-specific+phosphoprotein.+III.+Its+association+with+synaptic+vesicles+studied+in+a+highly+purified+synaptic+vesicle+preparation.">Synapsin I (protein I), a nerve terminal-specific phosphoprotein. III. Its association with synaptic vesicles studied in a highly purified synaptic vesicle preparation.</a> J Cell Biol. 96, 1374-1388 (1983).</li><li>Nagy, A., Baker, R. R., Morris, S. J., Whittaker, V. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+preparation+and+characterization+of+synaptic+vesicles+of+high+purity.">The preparation and characterization of synaptic vesicles of high purity.</a> Brain Res. 109, 285-309 (1976).</li><li>Cheng, D., 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=Relative+and+absolute+quantification+of+postsynaptic+density+proteome+isolated+from+rat+forebrain+and+cerebellum.">Relative and absolute quantification of postsynaptic density proteome isolated from rat forebrain and cerebellum.</a> Molecular &amp; Cellular Proteomics : MCP. 5, 1158-1170 (2006).</li><li>Sheng, M., Hoogenraad, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+postsynaptic+architecture+of+excitatory+synapses:+a+more+quantitative+view.">The postsynaptic architecture of excitatory synapses: a more quantitative view.</a> Annu Rev Biochem. 76, 823-847 (2007).</li><li>Sambrook, J., Fritsch, E. F., Maniatis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular cloning : a Laboratory Manual. , (1989).</li><li>Eggena, M., 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=Identification+of+histone+H1+as+a+cognate+antigen+of+the+ulcerative+colitis-associated+marker+antibody+pANCA.">Identification of histone H1 as a cognate antigen of the ulcerative colitis-associated marker antibody pANCA.</a> J Autoimmun. 14, 83-97 (2000).</li><li>Salahpour, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homodimerization+of+the+beta2-adrenergic+receptor+as+a+prerequisite+for+cell+surface+targeting.">Homodimerization of the beta2-adrenergic receptor as a prerequisite for cell surface targeting.</a> J Biol Chem. 279, 33390-33397 (2004).</li><li>Bernocco, 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=Sequential+detergent+fractionation+of+primary+neurons+for+proteomics+studies.">Sequential detergent fractionation of primary neurons for proteomics studies.</a> Proteomics. 8, 930-938 (2008).</li><li>Grunewald, T. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+localization+and+cytosolic+overexpression+of+LASP-1+correlates+with+tumor+size+and+nodal-positivity+of+human+breast+carcinoma.">Nuclear localization and cytosolic overexpression of LASP-1 correlates with tumor size and nodal-positivity of human breast carcinoma.</a> BMC Cancer. 7, 198 (2007).</li><li>Heimann, K., Percival, J. M., Weinberger, R., Gunning, P., Stow, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+isoforms+of+actin-binding+proteins+on+distinct+populations+of+Golgi-derived+vesicles.">Specific isoforms of actin-binding proteins on distinct populations of Golgi-derived vesicles.</a> J Biol Chem. 274, 10743-10750 (1999).</li><li>Neff, R. A., Gomez-Varela, D., Fernandes, C. C., Berg, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postsynaptic+scaffolds+for+nicotinic+receptors+on+neurons.">Postsynaptic scaffolds for nicotinic receptors on neurons.</a> Acta Pharmacol Sin. 30, 694-701 (2009).</li><li>Cella, N., Cornejo-Uribe, R. R., Montes, G. S., Hynes, N. E., Chammas, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+lysosomal-associated+membrane+protein+LAMP-1+is+a+novel+differentiation+marker+for+HC11+mouse+mammary+epithelial+cells.">The lysosomal-associated membrane protein LAMP-1 is a novel differentiation marker for HC11 mouse mammary epithelial cells.</a> Differentiation. 61, 113-120 (1996).</li><li>Otera, H., 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=Peroxisomal+targeting+signal+receptor+Pex5p+interacts+with+cargoes+and+import+machinery+components+in+a+spatiotemporally+differentiated+manner:+conserved+Pex5p+WXXXF/Y+motifs+are+critical+for+matrix+protein+import.">Peroxisomal targeting signal receptor Pex5p interacts with cargoes and import machinery components in a spatiotemporally differentiated manner: conserved Pex5p WXXXF/Y motifs are critical for matrix protein import.</a> Mol Cell Biol. 22, 1639-1655 (2002).</li><li>Goubaeva, F., 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=Cardiac+mitochondrial+connexin+43+regulates+apoptosis.">Cardiac mitochondrial connexin 43 regulates apoptosis.</a> Biochem Biophys Res Commun. 352, 97-103 (2007).</li><li>Lamers, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+S-100B,+neuron-specific+enolase+(NSE),+myelin+basic+protein+(MBP)+and+glial+fibrillary+acidic+protein+(GFAP)+in+cerebrospinal+fluid+(CSF)+and+blood+of+neurological+patients.">Protein S-100B, neuron-specific enolase (NSE), myelin basic protein (MBP) and glial fibrillary acidic protein (GFAP) in cerebrospinal fluid (CSF) and blood of neurological patients.</a> Brain Res Bull. 61, 261-264 (2003).</li><li>Arunachalam, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Munc18-1+is+critical+for+plasma+membrane+localization+of+syntaxin1+but+not+of+SNAP-25+in+PC12+cells.">Munc18-1 is critical for plasma membrane localization of syntaxin1 but not of SNAP-25 in PC12 cells.</a> Mol Biol Cell. 19, 722-734 (2008).</li><li>Brandstatter, J. H., Fletcher, E. L., Garner, C. C., Gundelfinger, E. D., Wassle, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+the+presynaptic+cytomatrix+protein+bassoon+among+ribbon+synapses+in+the+mammalian+retina.">Differential expression of the presynaptic cytomatrix protein bassoon among ribbon synapses in the mammalian retina.</a> Eur J Neurosci. 11, 3683-3693 (1999).</li></ol>

There are several steps in the procedure that are critical for a successful outcome. In step 3, it is important that a consistent degree of homogenization is achieved for each sample. When homogenizing tissue with the motor driven homogenizer, a constant speed is used not only for the rotation of the pestle, but also with the number of strokes. The incubation time during the osmotic shock should be precise, since extended homogenization or incubation in the hypotonic solution will lyse mitochondria and contaminate the SP...

Neurons communicate through synapses, and the quality of this communication is regulated to a large extent by alterations in the composition of proteins at the synapse. In particular, the proteins located in the post-synaptic density participate in neuronal communication by intimately scaffolding neurotransmitter receptors with their signal transduction systems1. Furthermore, lasting changes in the strength of synaptic efficacy are controlled by the addition or removal of receptors at the post-synaptic density1-6. Therefore, the isolation and quantification of synaptic proteins is a necessary and useful technique to gain insight into the ways that neurons respond to stimuli and alter synaptic efficacy7. This article describes a common technique to isolate synaptic proteins from rodent brain tissue by ultracentrifugation on discontinuous sucrose gradients. The synaptic plasma membrane fraction can be enriched and isolated based on its density in sucrose, which has been empirically determined to be similar to 1.2 M sucrose.
Depending on the biological question, subcellular fractions can be separated by continuous or discontinuous gradients of either sucrose or Percoll. Continuous gradients allow for the separation of proteins into multiple fractions; this can be particularly useful to demonstrate the co-localization of proteins within a given fraction8. However, the preparation of continuous gradients is more laborious and is unnecessary for many applications. Discontinuous gradients are comparatively easier to prepare and can be used to separate proteins into a few, generally-defined fractions. Discontinuous gradients that are composed of three sucrose layers of increasing molarity have been widely used to isolate proteins associated with the synaptic plasma membrane (SPM). This synaptic plasma membrane fraction can be further processed to the post-synaptic density fraction (PSD) by detergent treatment and isolation of the detergent-insoluble fraction.
When this process was first described in the 1960&#8217;s9,10, electron microscopy was used to demonstrate the organelles and membranes that roughly define the synaptic plasma membrane and post-synaptic density fractions9-14. These studies demonstrated the inclusion of pre- and post-synaptic membranes and synaptic vesicles in the SPM fraction; after detergent treatment primarily the electron-dense, post-synaptic densities were visible. In the procedure, a hypotonic shock is used to pinch off the synaptic processes from the cell body10. This step takes advantage of the fact that mitochondria are more resistant to osmotic shock and remain intact, and so they sediment at the bottom of the sucrose gradient (Figure 1).
Using this same enrichment technique, the SPM and PSD fractions were first biochemically defined by polyacrylamide gel electrophoresis and sequencing of the major protein components15-17. Subsequently western blot analysis has been used to detect and quantify the levels of synaptic proteins and further define these fractions (Figure 2). We have used this technique in our laboratories to quantify changes in the synaptic levels of the dopamine transporter that occur when the Slc6a3 locus is duplicated in mice18. We have also used this technique in NMDA receptor deficient mice to uncover synapse-specific reductions in proteins that are part of the DISC1 interactome19.
It is evident from western blot analysis that SPM fractions contain synaptic vesicle membrane proteins, endosome markers, mitochondrial proteins, membrane associated synthetic enzymes and signal transduction molecules, as well as integral components of the post-synaptic density and synaptic plasma membranes20-23. Even PSD fractions can have contamination with abundant mitochondrial proteins and it may be necessary to perform a second gradient sedimentation or additional purification steps to remove them13. Recently, quantitative mass spectrometry has provided a list of over 100 proteins in the post-synaptic density alone, as well as an indication of the relative abundance of these components24,25.

<table border="0" cellpadding="0" cellspacing="0" width="589">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">1M HEPES, pH 7.4</td>
			<td style="width:129px;">BioShop</td>
			<td style="width:111px;">HEP003.100</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">HEPES</td>
			<td style="width:129px;">BioShop</td>
			<td style="width:111px;">HEP001.500&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Sucrose&#160;</td>
			<td style="width:129px;">BioShop</td>
			<td style="width:111px;">SUC507.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">EDTA</td>
			<td style="width:129px;">BioBasic</td>
			<td style="width:111px;">EB0185</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">PMSF</td>
			<td style="width:129px;">BioShop</td>
			<td style="width:111px;">PMS123.5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Aprotinin</td>
			<td style="width:129px;">BioShop</td>
			<td style="width:111px;">APR600.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Leupeptin</td>
			<td style="width:129px;">BioShop</td>
			<td style="width:111px;">LEU001.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Pepstatin</td>
			<td style="width:129px;">BioShop</td>
			<td style="width:111px;">PEP605.5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Benzamidine</td>
			<td style="width:129px;">BioShop</td>
			<td style="width:111px;">BEN601.25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Sodium fluoride</td>
			<td style="width:129px;">BioShop</td>
			<td style="width:111px;">SFL001.100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Sodium pyrophosphate</td>
			<td style="width:129px;">BioShop</td>
			<td style="width:111px;">SPP310.100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:261px;">Sodium orthovanadate</td>
			<td style="width:129px;">BioShop</td>
			<td style="width:111px;">SOV664.10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">&#946;-glycerophosphate</td>
			<td style="width:129px;">BioShop</td>
			<td style="width:111px;">GYP001.10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Triton X-100</td>
			<td style="width:129px;">BioShop</td>
			<td style="width:111px;">TRX506.500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">18G x 1 &#189;&#8221; needle</td>
			<td style="width:129px;">BD</td>
			<td style="width:111px;">305196</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:261px;">1cc syringe</td>
			<td style="width:129px;">BD</td>
			<td style="width:111px;">309659</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:261px;"></td>
			<td style="width:129px;"></td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Name of Equipment</td>
			<td style="width:129px;">Company</td>
			<td>Catalog number</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">Glass Teflon homogenizer Kontes Duall 23</td>
			<td style="width:129px;">VWR</td>
			<td style="width:111px;">KT885450-0023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">IKA Model RW 16 Basic Stirrer</td>
			<td style="width:129px;">IKA Works</td>
			<td style="width:111px;">2572100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Sorvall SM-24 Fixed Angle Rotor</td>
			<td style="width:129px;">ThermoScientific</td>
			<td style="width:111px;">29017</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Sorvall RC 6 Plus Centrifuge</td>
			<td style="width:129px;">ThermoScientific</td>
			<td style="width:111px;">46910</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Thinwall Polyallomer Tubes (13.2mL)</td>
			<td style="width:129px;">Beckman Coulter</td>
			<td style="width:111px;">331372</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">SW 41 Ti Rotor Swinging Bucket</td>
			<td style="width:129px;">Beckman Coulter</td>
			<td style="width:111px;">331362</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Beckman L-80 Floor Ultracentrifuge</td>
			<td style="width:129px;">Beckman Coulter</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Thickwall Polycarbonate Tubes (3.5mL)</td>
			<td style="width:129px;">Beckman Coulter</td>
			<td style="width:111px;">349622</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">TLA-100.3 Fixed Angle Rotor</td>
			<td style="width:129px;">Beckman Coulter</td>
			<td style="width:111px;">349481</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:261px;">Beckman TL-100 Tabletop Ultracentrifuge</td>
			<td style="width:129px;">Beckman Coulter</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of synaptic plasma membrane and postsynaptic density proteins using a discontinuous sucrose gradient

Neuronal subcellular fractionation techniques allow the quantification of proteins that are trafficked to and from the synapse. As originally described in the late 1960&#8217;s, proteins associated with the synaptic plasma membrane can be isolated by ultracentrifugation on a sucrose density gradient. Once synaptic membranes are isolated, the macromolecular complex known as the post-synaptic density can be subsequently isolated due to its detergent insolubility. The techniques used to isolate synaptic plasma membranes and post-synaptic density proteins remain essentially the same after 40 years, and are widely used in current neuroscience research. This article details the fractionation of proteins associated with the synaptic plasma membrane and post-synaptic density using a discontinuous sucrose gradient. Resulting protein preparations are suitable for western blotting or 2D DIGE analysis.

The preparation of the sucrose density gradient should result in a clear separation of the three molar solutions of sucrose (0.8, 1.0, and 1.2 M sucrose). See Figure 3A for an example of the gradient before the protein sample is added. If the gradient is prepared too much in advance, or if it is prepared on a bench surface with vibration from other equipment, the gradient will be compromised and proper separation will not be achieved. If a clear separation of the three solutions is not visible, it is adv...

Watch this Scientific Journal Video about Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient at JoVE.com

Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient

This article details the enrichment of proteins associated with the synaptic plasma membrane by ultracentrifugation on a discontinuous sucrose gradient. The subsequent preparation of post-synaptic density proteins is also described. Protein preparations are suitable for western blotting or 2D DIGE analysis.

Neuronal subcellular fractionation techniques allow the quantification of proteins that are trafficked to and from the synapse. As originally described in ...

Research

JoVE Journal

Neuroscience

Studying Synaptic Vesicle Pools using Photoconversion of Styryl Dyes

The fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The ...

Membrane Potentials, Synaptic Responses, Neuronal Circuitry, Neuromodulation and Muscle Histology Using the Crayfish: Student Laboratory Exercises

The purpose of this report is to help develop an understanding of the effects caused by ion gradients across a biological membrane. Two aspects that ...

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

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 ...

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This ...

Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as ...

Deriving the Time Course of Glutamate Clearance with a Deconvolution Analysis of Astrocytic Transporter Currents

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the ...

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. ...