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;&#176;C and pre-cool all of the reagents and equipment prior to the start of the experiment. Label all tubes, including ultracentrifuge tubes, with permanent marker.</li>
</ol>
2. Dissection of the Appropriate Brain Region
<ol>
	<li>Sacrifice the animal by cervical dislocation or decapitation. House and euthanize animals in accordance with institutional and governmental policies on animal care.</li>
	<li>Rapidly remove the brain from the skull and place on an chilled dissection block.</li>
	<li>Dissect the appropriate brain region from fresh tissue and proceed to Step 3.</li>
</ol>
3. Tissue Homogenization with Motor Driven Glass-Teflon Homogenizer
<ol>
	<li>Place 50-100 mg (for SPM), or 100-200 mg (for PSD) of the dissected tissue in a labeled 13 ml polypropylene tube containing 4 ml of 0.32 M HEPES-buffered sucrose solution. Transfer the sample to a 15 ml tapered glass-Teflon tissue grinder/homogenizer, set the motor drive to 900 rpm (setting 7) and homogenize the sample with 12 strokes over a 30 sec period. Use a different glass-Teflon homogenizer between samples, or rinse the homogenizer with cold distilled water between samples and wipe dry with a Kimwipe. NOTE: Up to 4 g of tissue can be homogenized in 4 ml of 0.32 M HEPES-buffered sucrose solution, however it is advisable to use less tissue to ensure proper fractionation.</li>
	<li>Transfer the homogenized sample back to the same 13 ml polypropylene tube. Reserve 100 &#956;l of homogenate and store at -80 &#176;C for subsequent protein quantification and western blot analysis of the total protein fraction.</li>
</ol>
4. Low Speed Centrifugation to Remove the Nuclear Fraction (Yields Supernatant S1)
<ol>
	<li>Centrifuge the homogenate in a fixed angle rotor at 900 x g&#160;for 10 min at 4 &#176;C. Transfer the supernatant (S1) to a new, labeled 13 ml tube and resuspend the nuclear fraction pellet (P1) in 500 &#956;l of 0.32 M HEPES-buffered sucrose. NOTE: The (P1) fraction can be stored at -80 &#176;C and used for western blot analysis of the nuclear fraction.</li>
</ol>
5. Enrichment of the Crude Synaptosomal Fraction (Yields Pellet P2)
<ol>
	<li>Centrifuge the supernatant (S1) at 10,000 x g&#160;for 15 min at 4 &#176;C. Remove the supernatant (S2) and reserve 500 &#956;l in a microcentrifuge tube to store at -80 &#176;C for subsequent protein quantification and western blot analysis of the cytosolic/light membrane fraction. Resuspend the remaining crude synaptosomal fraction pellet (P2) in 1 ml of 0.32 M HEPES-buffered sucrose solution and then add another 3 ml of 0.32 M HEPES-buffered sucrose solution.</li>
	<li>Centrifuge at 10,000 x&#160;g for 15 min at 4 &#176;C. Remove the supernatant (S2&#8217;) and reserve 500 &#956;l in a microcentrifuge tube to store at -80 &#176;C for subsequent protein quantification and western blot analysis of the cytosolic/light membrane fraction. Save the washed crude synaptosomal pellet (P2&#8217;) in the polypropylene tube.</li>
</ol>
6. Lysing (Hypoosmotic Shock) of the Crude Synaptosomal Fraction
<ol>
	<li>Lyse the crude synaptosomal pellet (P2&#8217;) in the polypropylene tube by resuspending it in 1 ml of ddH2O. Add another 3 ml of ddH2O, transfer to a glass-Teflon tissue homogenizer, and homogenize by hand with 3 strokes. NOTE: It is important to perform this step as rapidly as possible and quickly proceed to step 6.2.</li>
	<li>Transfer samples from the homogenizer back into the same 13 ml polypropylene tube and rapidly adjust the sample back to 4 mM HEPES with 16 &#956;l&#160;of 1 M HEPES solution, invert to mix.</li>
	<li>Rotate samples at 4&#160;&#176;C for 30 min to ensure complete lysing.</li>
</ol>
7. Enrichment of the Synaptosomal Membrane Fraction (P3)
<ol>
	<li>Centrifuge at 25,000 x&#160;g for 20 min at 4 &#176;C. Remove the crude vesicular fraction supernatant (S3) and reserve it in a 5 ml tube to store at -80 &#176;C for subsequent protein quantification and western blot analysis. Resuspend the synaptosomal membrane fraction (P3) in 1 ml of 0.32 M HEPES-buffered sucrose solution.</li>
</ol>
8. Preparation of the Discontinuous Sucrose Gradient
<ol>
	<li>Pipet exactly 3.5 ml of 1.2 M HEPES-buffered sucrose solution and exactly 3.0 ml each of 1.0 M and 0.8 M HEPES-buffered sucrose solution into separate 6 ml snap cap tubes. NOTE: This is done to pre-measure the volumes of the buffers that are used to make the sucrose gradient. Exact measurement of the volumes is important to ensure that the resulting gradients will be balanced for ultracentrifugation.</li>
	<li>Using a glass Pasteur pipet and bulb, transfer 1.2 M HEPES-buffered sucrose solution to the 12 ml polyallomer ultracentrifuge tube. Carefully layer the 1.0 M HEPES-buffered sucrose solution on top of the 1.2 M HEPES-buffered sucrose solution. Finally, layer the 0.8 M HEPES-buffered sucrose solution on top of the 1.0 M HEPES-buffered sucrose solution. Use a fresh Pasteur pipet for each sucrose solution and take care not to disturb the sucrose gradient. Vibrations from a bench vortex or microcentrifuge will disturb the integrity of the gradient.</li>
	<li>Using a Pasteur pipet, layer the resuspended synaptosomal membrane fraction (P3) on top of the prepared discontinuous sucrose gradient. Ensure that the gradients are balanced by weighing them inside the bucket of the swinging bucket rotor. If the buckets are not balanced, use a P200 pipet to carefully add 0.32 M HEPES-buffered sucrose to the top of the gradient and balance the rotor buckets.</li>
</ol>
9. Fractionation of the Synaptic Plasma Membrane (SPM)
<ol>
	<li>Ultracentrifuge in a swinging bucket rotor at 150,000 x&#160;g for 2 hr at 4 &#176;C. Carefully remove the sucrose gradients from the ultracentrifuge buckets. Using an 18 G needle and a 1 ml syringe, puncture the tube at the bottom of the 1.0 M / 1.2 M HEPES-buffered sucrose solution interphase and withdraw the synaptic plasma membrane layer (SPM).</li>
	<li>Make note of the volume that each SPM sample occupies in the 1 ml syringe. Place the collected SPM layer in 3.5 ml thick-wall ultracentrifuge tubes, add exactly 2.5 volumes of 4 mM HEPES to adjust the sucrose concentration from 1.2 M to 0.32 M. Once each sample has been adjusted to 0.32 M sucrose, balance the tubes with 0.32 M HEPES-buffered sucrose solution.</li>
	<li>Ultracentrifuge in a fixed angle rotor at 200,000 x&#160;g for 30 min at 4 &#176;C. Remove and discard the supernatant. Resuspend the synaptic plasma membrane pellet (SPM) in 300 &#956;l of 50 mM HEPES / 2 mM EDTA solution. Store samples at -80 &#176;C until used for western blotting.</li>
</ol>
10. Preparation of the Postsynaptic Density Fraction (PSD)
<ol>
	<li>Thaw SPM samples on ice.</li>
	<li>Make a solution of 0.54% Triton X-100 in 50 mM HEPES&#160;/&#160;2 mM EDTA solution.</li>
	<li>In a 5 ml polystyrene tube, combine 2.7 ml of Triton X-100/HEPES/EDTA solution and 300 &#956;l SPM fraction and rotate the sample for 15 min at 4 &#176;C</li>
	<li>Transfer samples to 3.5 ml thick-wall ultracentrifuge tubes and centrifuge samples at 32,000 x&#160;g for 20 min at 4 &#176;C.</li>
	<li>Reserve the supernatant (Triton X-100 soluble fraction [TS]) and store at -80&#160;&#176;C. NOTE: At this stage, the pellet represents the &#8220;PSD-1T&#8221; fraction. A second treatment with detergent will produce a &#8220;PSD-2T&#8221; fraction. If a PSD-1T fraction is desired, proceed to step 10.6.
	<ol>
		<li>To produce a PSD-2T fraction, resuspend the pellet in 3 ml of 0.5% Triton X-100 in 50 mM HEPES&#160;/ 2 mM EDTA solution. Rotate samples for 15 min at 4 &#176;C. Centrifuge samples at 32,000 x&#160;g for 20 min at 4 &#176;C and proceed to step 10.6.</li>
	</ol>
	</li>
	<li>Discard the supernatant and resuspend the postsynaptic (PSD) pellet in 50 mM HEPES / 2 mM EDTA solution. NOTE: The resuspension volume may depend on the size of the pellet; a volume of 50-75 &#956;l is recommended. Because the PSD fraction is a detergent-insoluble fraction, it is often necessary to add 1-2 &#956;l of 0.5% SDS to enable complete resuspension of protein. Store samples at -80 &#176;C until used for western blotting.</li>
</ol>

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<li>Arunachalam, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Munc18-1+is+critical+for+plasma+membrane+localization+of+syntaxin1+but+not+of+SNAP-25+in+PC12+cells%5BTitle%5D)+AND+%22Mol+Biol+Cell%22%5BJournal%5D)">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/pubmed?term=((Differential+expression+of+the+presynaptic+cytomatrix+protein+bassoon+among+ribbon+synapses+in+the+mammalian+retina%5BTitle%5D)+AND+%22Eur+J+Neurosci%22%5BJournal%5D)">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>

The authors have no conflicts of interest to disclose.

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 SPM sample.
Another critical step is in the preparation of reagents, especially the sucrose solutions; if the molarity of the sucrose solution is incorrect the procedure will not work. Therefore, sucrose solutions should be prepared by weighing sucrose, dissolving in 4 mM HEPES solution then adding 4 mM HEPES to the precise final volume. Troubleshooting tip: build a test gradient with the prepared sucrose solutions to ensure that the gradient is properly assembled. Add varying concentrations of bromophenol blue (for example, final concentration of 0.005%-0.02% w/v) to the two of the three sucrose solutions to assist in the visualization of three distinct layers.
It is important to limit the amount of time between when the gradient is assembled and when the tubes are centrifuged. Normal diffusion will destroy the gradient over time, and this is accelerated if there is vibration on the bench where the gradient is stored. Pre-pipetting of solutions into individual aliquots allows for precise volumes to be pipetted with a glass Pasteur pipet. This also decreases the time between gradient assembly and ultracentrifugation. Troubleshooting tip: build a &#8220;test gradient&#8221; with bromophenol blue dye as described above at the same time as the sucrose gradients for the experiment. The integrity of the gradient can thus be monitored over time. The test gradient will serve as an indicator of diffusion if the gradients are assembled too far in advance or are exposed to vibration.
Finally, it is important to collect the SPM sample in as small a volume as possible, since it is necessary to subsequently dilute the sample with 2.5 volumes of water. Ideally the sample should be collected in a volume of 0.4-0.7 ml&#160;using a 1 cc syringe.&#160;It is advisable to build the discontinuous gradients during the final 20 min spin in step 7 to limit time between gradient assembly and ultracentrifugation.
The protocol includes the recommendation to reserve all of the different fractions and store these at -80&#160;&#176;C for subsequent analysis. We have demonstrated the enrichment of post-synaptic proteins in Figure 2 in only a few of these fractions. However, it is advisable to measure the levels of a protein-of-interest in each of the fractions to understand its distribution. Comparing the distribution of a protein in the fractions with other subcellular marker proteins will also help to confirm that the experimental conditions have achieved the desired fractionation. Table 2 summarizes commonly-used protein markers that can be used as indicators for the stages of fractionation8,27-38.
Utilization of the discontinuous sucrose gradient is widely used to enrich synaptic plasma membranes. This method has the advantage over continuous Percoll gradients in that discontinuous sucrose gradients are easier to make and do not require specialized equipment. However, this method may not be sufficient to precisely localize a protein of interest, and for these applications a Percoll gradient may be preferred.

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><tbody><tr><td>1 M HEPES, pH 7.4</td><td>BioShop</td><td>HEP003.100</td><td></td></tr><tr><td>HEPES</td><td>BioShop</td><td>HEP001.500&amp;nbsp;</td><td></td></tr><tr><td>Sucrose&amp;nbsp;</td><td>BioShop</td><td>SUC507.1</td><td></td></tr><tr><td>EDTA</td><td>BioBasic</td><td>EB0185</td><td></td></tr><tr><td>PMSF</td><td>BioShop</td><td>PMS123.5</td><td></td></tr><tr><td>Aprotinin</td><td>BioShop</td><td>APR600.1</td><td></td></tr><tr><td>Leupeptin</td><td>BioShop</td><td>LEU001.1</td><td></td></tr><tr><td>Pepstatin</td><td>BioShop</td><td>PEP605.5</td><td></td></tr><tr><td>Benzamidine</td><td>BioShop</td><td>BEN601.25</td><td></td></tr><tr><td>Sodium fluoride</td><td>BioShop</td><td>SFL001.100</td><td></td></tr><tr><td>Sodium pyrophosphate</td><td>BioShop</td><td>SPP310.100</td><td></td></tr><tr><td>Sodium orthovanadate</td><td>BioShop</td><td>SOV664.10</td><td></td></tr><tr><td>&amp;beta;-glycerophosphate</td><td>BioShop</td><td>GYP001.10</td><td></td></tr><tr><td>Triton X-100</td><td>BioShop</td><td>TRX506.500</td><td></td></tr><tr><td>18 G x 1 &amp;frac12;&amp;rdquo; needle</td><td>BD</td><td>305196</td><td></td></tr><tr><td>1 cc syringe</td><td>BD</td><td>309659</td><td></td></tr><tr><td>Glass Teflon homogenizer Kontes Duall 23</td><td>VWR</td><td>KT885450-0023</td><td></td></tr><tr><td>IKA Model RW 16 basic stirrer</td><td>IKA Works</td><td>2572100</td><td></td></tr><tr><td>Sorvall SM-24 fixed angle rotor</td><td>ThermoScientific</td><td>29017</td><td></td></tr><tr><td>Sorvall RC 6 Plus centrifuge</td><td>ThermoScientific</td><td>46910</td><td></td></tr><tr><td>Thinwall polyallomer tubes (13.2 ml)</td><td>Beckman Coulter</td><td>331372</td><td></td></tr><tr><td>SW 41 Ti rotor swinging bucket</td><td>Beckman Coulter</td><td>331362</td><td></td></tr><tr><td>Beckman L-80 floor ultracentrifuge</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>Thickwall polycarbonate tubes (3.5 ml)</td><td>Beckman Coulter</td><td>349622</td><td></td></tr><tr><td>TLA-100.3 fixed angle rotor</td><td>Beckman Coulter</td><td>349481</td><td></td></tr><tr><td>Beckman TL-100 tabletop ultracentrifuge</td><td>Beckman Coulter</td><td></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 advisable to make a new gradient before adding the protein sample. See Figure 3B for an example of the sucrose gradient when the protein sample is added (black arrow).
Following the centrifugation, there should be clear bands of protein fractions at each interphase (0.32/0.8, 0.8/1.0, 1.0/1.2). In addition a pellet should be visible at the bottom of the tube. See Figure 3C for an example of the fraction bands after ultracentrifugation. If fractions are only diffusely present at the interphase, it is likely that the gradient was compromised and further processing is not advised.
Western blotting of total, SPM and PSD1T protein samples from mouse striatum is demonstrated in Figure 2. The enrichment of synaptic proteins like GluR1, PSD95, and CaMKII, can be visualized by loading a constant amount of protein (10 &#956;g) from total, SPM, and PSD fractions. Perisynaptic proteins like the dopamine transporter (DAT) are enriched in the SPM fraction, but are eliminated in subsequent detergent steps of the PSD enrichment process.
To ensure equivalent loading, protein levels can be visualized on the western blot membrane by staining with Ponceau Red26. Antibody-based &#8220;loading controls&#8221; are often reported in the literature, but this may be a misnomer since synaptic proteins may be differentially regulated in experimental treatment groups. If an antibody-based &#8220;loading control&#8221; is desired, it is advisable to first determine equivalent protein loading by Ponceau staining, and then test several reference proteins to identify those that do not change in the different experimental groups.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51896/51896fig1highres.jpg" width="500" /> 
Figure 1. Workflow of subcellular fractionation to enrich SPM and PSD protein fractions.
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51896/51896fig2highres.jpg" width="500" /> 
Figure 2. Representative western blot of total, SPM, and PSD1T protein from mouse striatum. 10 &#956;g of protein extract were resolved on SDS-denaturing, 10% acrylamide gels, and transferred to PVDF membranes. Subsequently these blots were incubated with primary antibodies for &#945;CaMKII, GluR1, PSD-93, and PSD-95, which are all components of the post-synaptic density. Accordingly, these proteins are enriched in the SPM and PSD fractions. Alternately, blots were incubated with a primary antibody for DAT, which is located in presynaptic membranes in the striatum. While DAT is enriched in the SPM fraction, it is not part of the PSD complex, and immunoreactivity is not observed in the PSD fraction. Antibodies against actin or sodium/potassium ATPase (Na/K pump) can be used as loading controls, although some experimental conditions may affect the levels of these proteins as well and so the appropriate loading control must be determined empirically.
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/51896/51896fig3highres.jpg" width="500" /> 
Figure 3. Representative examples of sucrose gradient before and after ultracentrifugation. A.&#160;After the gradient is prepared, there should be three phases that are visible when the tube is held up to the light. The white arrows indicate the interphase between 0.8/1.0 M sucrose solutions and 1.0/1.2 M sucrose solutions. B. When the sample is loaded on top of the gradient, it is in a 0.32 M sucrose solution and will rest on top of the 0.8 M sucrose layer. C. After centrifugation, the homogenate will separate into several fractions. The fraction floating at the interphase of 0.8/1.0 M sucrose solutions will be enriched in myelin membranes. The fraction at the interphase of 1.0/1.2 M sucrose solutions represents the SPM fraction that is collected with a needle and syringe. The pellet at the bottom of the tube is enriched for mitochondrial proteins.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Solutions</td>
			<td>HEPES-buffered sucrose</td>
			<td>Protease inhibitors</td>
			<td>*Phophatase inhibitors</td>
		</tr>
		<tr>
			<td>1 M HEPES pH 7.4&#160;</td>
			<td>0.32 M sucrose in 4 mM HEPES (pH 7.4)</td>
			<td>0.25 mM PMSF (stock 250 mM in ethanol, 1,000x)&#160;</td>
			<td>10 mM Sodium Fluoride (stock 500 mM, 50x)</td>
		</tr>
		<tr>
			<td>4 mM HEPES pH 7.4</td>
			<td>0.8 M sucrose in 4 mM HEPES (pH 7.4)</td>
			<td>1.5 &#956;g/ml Aprotinin (stock 1.5 mg/ml, 1,000x)</td>
			<td>2.5 mM Sodium Pyrophosphate (stock 250 mM, 100x)</td>
		</tr>
		<tr>
			<td>ddH2O</td>
			<td>1.0 M sucrose in 4 mM HEPES (pH 7.4)</td>
			<td>10 &#956;g/ml Leupeptin (stock 10 mg/ml, 1,000x)</td>
			<td>1.0 mM b-glycerophosphate (stock 200 mM, 200x)</td>
		</tr>
		<tr>
			<td>50 mM HEPES (pH 7.4) 2 mM EDTA</td>
			<td>1.2 M sucrose in 4 mM HEPES (pH 7.4)</td>
			<td>0.1 mg/ml Benzamidine (stock 100 mg/ml, 1,000x)</td>
			<td>5.0 mM Sodium Orthovanadate (stock 500 mM, 100x)</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>10 &#956;g/ml Pepstatin (stock 5 mg/ml in ethanol, 500x)</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1. Required Reagents
<table border="0" cellpadding="0" cellspacing="0" width="614">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:88px;">Fraction&#160;</td>
			<td style="width:191px;">Protein fraction</td>
			<td style="width:335px;">Protein marker</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">P1</td>
			<td>nuclear&#160;</td>
			<td>histone H127</td>
		</tr>
		<tr height="72">
			<td height="72" style="height:72px;">S1</td>
			<td>cytosol/membranes</td>
			<td style="width:335px;">calnexin28 (endoplasmic reticulum), a-tubulin29 (cytoskeleton), GAPDH30 (cytosol), 58K golgi protein31 (Golgi apparatus)</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">P2 and P2&#39;</td>
			<td>crude synaptosomes</td>
			<td style="width:335px;">AMPA and NMDA receptor subunits32</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;">S2 and S2&#39;</td>
			<td>cytosol/light membranes</td>
			<td style="width:335px;">nNOS129 (cytosol), GAPDH (cytosol), LAMP133 (lysosome), PEX1434 (peroxisome)</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;">P3</td>
			<td>synaptosome/mitochondria</td>
			<td style="width:335px;">VDAC35 (mitochondria), AMPA and NMDA receptor subunits (synaptosome)</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">S3</td>
			<td>synaptic vesicle</td>
			<td style="width:335px;">SV28, synaptophysin8</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">0.8 M/1.0 M</td>
			<td>myelin</td>
			<td style="width:335px;">myelin basic protein36</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;">1.0 M/1.2 M</td>
			<td>SPM</td>
			<td style="width:335px;">AMPA and NMDA receptor subunits, presynaptic marker (SNAP258), neurexin32, neuroligin32</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">1.2 M P</td>
			<td>mitochondria</td>
			<td style="width:335px;">VDAC35</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">PSD (1T)</td>
			<td>PSD</td>
			<td style="width:335px;">PSD9332, PSD9532, AMPA and NMDA receptor subunits, neuroligin, PICK132, CaMKII32</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">TS</td>
			<td>presynaptic membranes</td>
			<td style="width:335px;">SNAP25, Munc1837, Bassoon38, neurexin</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;">PSD-2T</td>
			<td>enriched PSD</td>
			<td style="width:335px;">PSD95, AMPA and NMDA receptor subunits, PICK1, CaMKII</td>
		</tr>
	</tbody>
</table>
Table 2. List of protein markers to distinguish subcellular fractions

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 ...

preparation-synaptic-plasma-membrane-postsynaptic-density-proteins

Nanyang Technological University

Research

JoVE Journal

Neuroscience

31.2K Views.  University of Toronto.  Dit artikel beschrijft de verrijking van eiwitten geassocieerd met de synaptische plasmamembraan door ultracentrifugatie op een discontinue sucrose gradiënt. De verdere uitwerking van postsynaptische dichtheid eiwit wordt ook beschreven. Eiwitpreparaten zijn geschikt voor western blotting of 2D DIGE analyse. 

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

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.

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.

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

Biochemistry

Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription reflect changes in protein synthesis, but many exceptions have been observed. Recently, a technique called ribosome profiling (or Ribo-Seq) has emerged as a powerful method that allows identification, with high accuracy, which regions of mRNA are translated into proteins and quantification of translation at the genome-wide level. Here, we present a generalized protocol for genome-wide quantification of translation using Ribo-Seq in budding yeast. In addition, combining Ribo-Seq data with mRNA abundance measurements allows us to simultaneously quantify translation efficiency of thousands of mRNA transcripts in the same sample and compare changes in these parameters in response to experimental manipulations or in different physiological states. We describe a detailed protocol for generation of ribosome footprints using nuclease digestion, isolation of intact ribosome-footprint complexes via sucrose gradient fractionation, and preparation of DNA libraries for deep sequencing along with appropriate quality controls necessary to ensure accurate analysis of in vivo translation.

Translational regulation plays an important role in the control of protein abundance. Here, we describe a high-throughput method for quantitative analysis of translation in the budding yeast Saccharomyces cerevisiae.

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription ...

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 ...

Density Gradient Ultracentrifugation for Investigating Endocytic Recycling in Mammalian Cells

Endosomal trafficking is an essential cellular process that regulates a broad range of biological events. Proteins are internalized from the plasma membrane and then transported to the early endosomes. The internalized proteins could be transited to the lysosome for degradation or recycled back to the plasma membrane. A robust endocytic recycling pathway is required to balance the removal of membrane materials from endocytosis. Various proteins are reported to regulate the pathway, including ADP-ribosylation factor 6 (ARF6). Density gradient ultracentrifugation is a classical method for cell fractionation. After the centrifugation, organelles are sedimented at their isopycnic surface. The fractions are collected and used for other downstream applications. Described here is a protocol to obtain a recycling endosome-containing fraction from transfected mammalian cells using density gradient ultracentrifugation. The isolated fractions were subjected to standard Western blotting for analyzing their protein contents. By employing this method, we identified that the plasma membrane targeting of engulfment and cell motility 1 (ELMO1), a Ras-related C3 botulinum toxin substrate 1 (Rac1) guanine nucleotide exchange factor, is through ARF6-mediated endocytic recycling.

This paper aims to present a protocol for preparing recycling endosomes from mammalian cells using sucrose density gradient ultracentrifugation.

Endosomal trafficking is an essential cellular process that regulates a broad range of biological events. Proteins are internalized from the plasma ...

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 ...

Isolation of Mitochondrial Contact Sites

An Improved Method to Isolate Mitochondrial Contact Sites

Mitochondria are present in virtually all eukaryotic cells and perform essential functions that go far beyond energy production, for instance, the synthesis of iron-sulfur clusters, lipids, or proteins, Ca2+ buffering, and the induction of apoptosis. Likewise, mitochondrial dysfunction results in severe human diseases such as cancer, diabetes, and neurodegeneration. In order to perform these functions, mitochondria have to communicate with the rest of the cell across their envelope, which consists of two membranes. Therefore, these two membranes have to interact constantly. Proteinaceous contact sites between the mitochondrial inner and outer membranes are essential in this respect. So far, several contact sites have been identified. In the method described here, Saccharomyces cerevisiae mitochondria are used to isolate contact sites and, thus, identify candidates that qualify for contact site proteins. We used this method to identify the mitochondrial contact site and cristae organizing system (MICOS) complex, one of the major contact site-forming complexes in the mitochondrial inner membrane, which is conserved from yeast to humans. Recently, we further improved this method to identify a novel contact site consisting of Cqd1 and the Por1-Om14 complex.

Author Spotlight: Unveiling Mitochondrial Contact Sites and Architectural Insights 

Mitochondrial contact sites are protein complexes that interact with mitochondrial inner and outer membrane proteins. These sites are essential for the communication between the mitochondrial membranes and, thus, between the cytosol and the mitochondrial matrix. Here, we describe a method to identify candidates qualifying for this specific class of proteins.

Mitochondria are present in virtually all eukaryotic cells and perform essential functions that go far beyond energy production, for instance, the ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

High Resolution Quantitative Synaptic Proteome Profiling of Mouse Brain Regions After Auditory Discrimination Learning

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory discrimination learning in mice is described. In this learning paradigm, mice are trained in a shuttle box Go/NoGo-task to discriminate between rising and falling frequency-modulated tones in order to avoid a mild electric foot-shock. The protocol involves the enrichment of synaptosomes from four brain areas, namely the auditory cortex, frontal cortex, hippocampus, and striatum, at different stages of training. Synaptic protein expression patterns obtained from trained mice are compared to na&#239;ve controls using a proteomic approach. To achieve sufficient analytical depth, samples are fractionated in three different ways prior to mass spectrometry, namely 1D SDS-PAGE/in-gel digestion, in-solution digestion and phospho-peptide enrichment. 
High-resolution proteomic analysis on a mass spectrometer and label-free quantification are used to examine synaptic protein profiles in phospho-peptide-depleted and phospho-peptide-enriched fractions of synaptosomal protein samples. A commercial software package is utilized to reveal proteins and phospho-peptides with significantly regulated relative synaptic abundance levels (trained/na&#239;ve controls). Common and differential regulation modes for the synaptic proteome in the investigated brain regions of mice after training were observed. Subsequently, meta-analyses utilizing several databases are employed to identify underlying cellular functions and biological pathways.

The identification of molecules and pathways controlling synaptic plasticity and memory is still a major challenge in neuroscience. Here, a workflow is described addressing the relative quantification of synaptic proteins supposedly involved in the molecular reorganization of synapses during learning and memory consolidation in an auditory learning paradigm.

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory ...

Subcellular Fractionation for the Isolation of Synaptic Components from the Murine Brain

Synaptic terminals are the primary sites of neuronal communication. Synaptic dysfunction is a hallmark of many neuropsychiatric and neurological disorders. The characterization of synaptic sub-compartments by biochemical isolation is, therefore, a powerful method to elucidate the molecular bases of synaptic processes, both in health and disease. This protocol describes the isolation of synaptic terminals and synaptic sub-compartments from mouse brains by subcellular fractionation. First, sealed synaptic terminal structures, known as synaptosomes, are isolated following brain tissue homogenization. Synaptosomes are neuronal pre- and post-synaptic compartments with pinched-off and sealed membranes. These structures retain a metabolically active state and are valuable for studying synaptic structure and function. The synaptosomes are then subjected to hypotonic lysis and ultracentrifugation to obtain synaptic sub-compartments enriched for synaptic vesicles, synaptic cytosol, and synaptic plasma membrane. Fraction purity is confirmed by electron microscopy and biochemical enrichment analysis for proteins specific to sub-synaptic compartments. The presented method is a straightforward and valuable tool for studying the structural and functional characteristics of the synapse and the molecular etiology of various brain disorders.

This protocol presents a robust, detailed method to obtain highly pure synaptosomes, synaptic vesicles, and other synaptic fractions from the mouse brain. This method enables the evaluation of synaptic processes, including the biochemical analysis of protein localization and function with compartmental resolution.

Synaptic terminals are the primary sites of neuronal communication. Synaptic dysfunction is a hallmark of many neuropsychiatric and neurological ...

An Easy Method for Plant Polysome Profiling

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the analysis of translational regulation. The method described here is an easy and economical way for fractionating polysomes from various plant tissues. A sucrose gradient is made without the need for a gradient maker by sequentially freezing each layer. Cytosolic extracts are then prepared in a buffer containing cycloheximide and chloramphenicol to immobilize the cytosolic and chloroplastic ribosomes to mRNA and are loaded onto the sucrose gradient. After centrifugation, six fractions are directly collected from the bottom to the top of the gradient, without piercing the ultracentrifugation tube. During collection, the absorbance at 260 nm is read continuously to generate a polysome profile that gives a snapshot of global translational activity. Fractions are then pooled to prepare three different mRNA populations: the polysomes, mRNAs bound to several ribosomes; the monosomes, mRNAs bound to one ribosome; and mRNAs that are not bound to ribosomes. mRNAs are then extracted. This protocol has been validated for different plants and tissues including Arabidopsis thaliana seedlings and adult plants, Nicotiana benthamiana, Solanum lycopersicum, and Oryza sativa leaves.

This protocol describes an easy method to extract and fractionate transcripts from plant tissues on the basis of the number of bound ribosomes. It allows a global estimate of translation activity and the determination of the translational status of specific mRNAs.

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the ...

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

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