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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.&#160;&#160;
1. Preparation of mouse brain membranes
<ol>
	<li>Decapitate mouse using a guillotine apparatus and dissect brain out rapidly (in &#60;1 min, if possible, to minimize palmitoylation changes that may occur during dissection procedure). Homogenize immediately in 10 mL of homogenization buffer (Table 1) in a glass homogenizer (~12 strokes) on ice.</li>
	<li>Centrifuge lysates for 15 min at 1,400 x&#160;g&#160;at 4 &#176;C. Transfer the supernatant to a new tube on ice. Resuspend the pellet (~6 strokes) in an equal volume of homogenization buffer and centrifuge at 710 x&#160;g&#160;for 10 min at 4 &#176;C.</li>
	<li>Combine the supernatants from step 1.2 and centrifuge at 40,000 x&#160;g&#160;for 20 min at 4 &#176;C.</li>
	<li>Discard the supernatant (cytosolic fraction) and resuspend the pelleted membrane (P2) fraction in homogenization buffer. 
	NOTE: Samples can be flash frozen and stored at -80 &#176;C for later use.</li>
	<li>Perform a bicinchoninic acid (BCA) assay to quantitate protein levels. For the Acyl-PEGyl exchange gel-shift (APEGS) assay, begin with ~100&#8722;200 mg protein (maximum 250 mg) in a volume of 470 &#181;L of buffer A (Table 1) in a 1.5 mL tube. Sonicate and centrifuge at &#62;13,000 x&#160;g&#160;for 10 min at 25 &#176;C to remove insoluble protein. Transfer solubilized protein to a new 1.5 mL tube.</li>
</ol>
2. Acyl-PEGyl exchange gel-shift (APEGS) assay
<ol>
	<li>Disrupt disulfide bonds and block free cysteines.
	<ol>
		<li>Disrupt disulfide bonds by incubating in 25 mM tris(2-carboxyethyl)phosphine (TCEP; 25 &#181;L, added from a stock solution;&#160;Table 1) for 60 min at 55 &#176;C.</li>
		<li>Block free cysteines with 50 mM N-ethylmaleimide (NEM; 12.5 &#181;L, added from a stock solution;&#160;Table 1) at room temperature for 3 h. 
		NOTE: The reaction can be extended overnight.</li>
	</ol>
	</li>
	<li>Perform chloroform methanol (CM) precipitation.
	<ol>
		<li>Transfer the protein solution from 1.5 mL tube to a polypropylene or glass tube that can be centrifuged in a swinging bucket rotor at modest speed. A 5 mL tube is ideal. Add four volumes (2 mL) of methanol (MeOH) and vortex briefly.</li>
		<li>Add two volumes (1 mL) of chloroform and vortex briefly.</li>
		<li>Add three volumes (1.5 mL) of deionized water (dH2O) and vortex briefly.</li>
		<li>Centrifuge the samples at 3,000 x&#160;g&#160;for 30 min at 25 &#176;C in a swinging bucket rotor.</li>
		<li>Carefully remove and discard the upper phase.</li>
		<li>Add 3 volumes (1.5 mL) of MeOH and mix gently but thoroughly, being careful not to fragment the opaque protein pancake.</li>
		<li>Centrifuge at 3,000 x&#160;g&#160;for 10 min at 25 &#176;C in a swinging bucket rotor.</li>
		<li>Using a glass serological pipet, remove as much of the top phase as possible without disturbing the protein pancake.</li>
		<li>Carefully rinse the pellet with 1 mL of MeOH.</li>
		<li>Air dry the pellet for at least 10 min. 
		NOTE: Dried pellet can be stored at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Perform cleavage of palmitoyl-thioester linkages with hydroxylamine (NH2OH, HAM).
	<ol>
		<li>Resuspend the protein precipitate in 125 &#181;L of buffer A and sonicate briefly. Centrifuge at &#62;13,000 x&#160;g&#160;for 10 min at 25 &#176;C to remove insoluble material. Transfer solubilized protein to a new 1.5 mL tube.</li>
		<li>Add 375 &#181;L of buffer H (HAM+;&#160;Table 1) or buffer T (HAM-;&#160;Table 1) and incubate samples for 60 min at 25 &#176;C.</li>
	</ol>
	</li>
	<li>Repeat the CM precipitation described in step 2.2. 
	NOTE: Dried pellet can be stored at -20 &#176;C.</li>
	<li>Add mPEG to unprotected cysteines.
	<ol>
		<li>Resuspend pellet in 100 &#181;L of buffer A containing 10 mM TCEP. Transfer to a 1.5 mL tube. 
		NOTE: It is normal for significant loss of protein to have occurred during CM precipitation steps. All subsequent steps will be performed in a 1.5 mL tube, constraining the volume of protein to a maximum of ~120&#8722;130 &#181;L. This will reduce protein loss during the final CM precipitation.</li>
		<li>Add 20 mM mPEG-5k (25 &#181;L, added from a stock solution;&#160;Table 1) to protein sample and mix by pipetting. Incubate for 60 min at 25 &#176;C with end-over-end rotation.</li>
		<li>To remove unincorporated mPEG-5k, perform CM precipitation as described in step 2.2 using these adjusted volumes: 4 volumes of MeOH (500 &#181;L), 2 volumes of chloroform (250 &#181;L), and 3 volumes of dH2O (375 &#181;L). Centrifuge at &#62;13,000 x&#160;g&#160;for 10 min at 25 &#176;C.</li>
		<li>Carefully remove the upper phase as before, avoiding the thick, flocculent pancake. Add 1 mL of MeOH and mix gently but thoroughly. Centrifuge at &#62;13,000 x&#160;g&#160;for 10 min at 25 &#176;C.</li>
		<li>Carefully remove the supernatant and rinse the pellet with 1 mL of MeOH. Centrifuge at &#62;13,000 x&#160;g&#160;for 10 min at 25 &#176;C. Air dry pellet. 
		NOTE: Dried pellet can be stored at -20 &#176;C.</li>
		<li>Resuspend the dried pellet in 50 &#181;L of buffer A (without TCEP or any reducing agent).</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Hydroxylamine (HAM)&#160;</td>
			<td>ThermoFisher&#160;</td>
			<td>26103</td>
			<td></td>
		</tr>
		<tr>
			<td>Methoxy-PEG-(CH2)3NHCO(CH2)2-MAL (mPEG)</td>
			<td>NOF</td>
			<td>ME-050MA</td>
			<td>MW ~5000 kDa</td>
		</tr>
		<tr>
			<td>Microfuge</td>
			<td>Eppendorf</td>
			<td>5415R</td>
			<td>or equivalent equipment</td>
		</tr>
		<tr>
			<td>N-ethylmalemide (NEM)</td>
			<td>Calbiochem</td>
			<td>34115</td>
			<td>Highly toxic</td>
		</tr>
		<tr>
			<td>Optical imager for densitometry</td>
			<td>Azure Biosystems</td>
			<td>Sapphire Biomolecular Imager</td>
			<td>or equivalent equipment</td>
		</tr>
		<tr>
			<td>Polypropylene tubes with cap</td>
			<td>Fisher Scientific</td>
			<td>14-956-1D</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipets (glass)</td>
			<td>Fisher Scientific</td>
			<td>13-678-27D</td>
			<td></td>
		</tr>
		<tr>
			<td>Table top centrifuge</td>
			<td>Beckman</td>
			<td>Allegra X-15R</td>
			<td>or equivalent equipment</td>
		</tr>
		<tr>
			<td>Tris(2-carboxyethyl)phosphinehydrochloride (TCEP)</td>
			<td>EMD Millipore</td>
			<td>580560</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessing the palmitoylation state of cell membrane proteins from mouse brain tissue

Source: Speca, D. J., et al. <a href="https://app.jove.com/v/61018">Acyl-PEGyl Exchange Gel Shift Assay for Quantitative Determination of Palmitoylation of Brain Membrane Proteins.</a> J. Vis. Exp. (2020).
The video demonstrates the analysis of the palmitoylation state of cell membrane proteins isolated from mouse brain tissue. The process involves exposing the non-palmitoylated cysteines and blocking them, then using a polymer that binds to the palmitoylated cysteines, aiding in assessing the palmitoylation state of the isolated proteins.

Table 1: Solutions used for APEGS assay. Homogenization buffer and buffer A can be prepared ahead of time; however, add protease inhibitors and phenylmethylsulfonyl fluoride (PMSF) immediately before use. All other solutions should be prepared fresh.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Buffer</td>
			<td>Components</td>
			<td>Working conc.</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Homogenization</td>
			<td>0.32 M sucrose, 1 mM Tris pH 7.2, 1 mM MgCl2</td>
			<td></td>
			<td>Add PMSF and protease inhibitors immediately before use.</td>
		</tr>
		<tr>
			<td>Buffer A</td>
			<td>PBS pH 7.2, 5 mM EDTA, 4% SDS (w/v)</td>
			<td></td>
			<td>Add PMSF and protease inhibitors immediately before use.</td>
		</tr>
		<tr>
			<td>TCEP solution</td>
			<td>500 mM TCEP in ddH2O (pH 7.2)</td>
			<td>25 mM</td>
			<td>Add 10 N NaOH to increase pH.</td>
		</tr>
		<tr>
			<td>NEM solution</td>
			<td>2.0 M NEM in 100% ethanol</td>
			<td>50 mM</td>
			<td>Prepare fresh.</td>
		</tr>
		<tr>
			<td>Buffer H (HAM+)</td>
			<td>1.33 M HAM, pH 7.0, 5 mM EDTA, 0.2% Triton X-100 (w/v)</td>
			<td>1.0 M</td>
			<td>Prepare fresh.</td>
		</tr>
		<tr>
			<td>Buffer T (HAM-)</td>
			<td>1.33 M Tris-HCl, pH 7.0, 5 mM EDTA, 0.2% Triton X-100 (w/v)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>mPEG-5k</td>
			<td>100 mM mPEG-5k in ddH2O</td>
			<td>20 mM</td>
			<td>Mix well. Highly viscous.</td>
		</tr>
	</tbody>
</table>

Assessing the Palmitoylation State of Cell Membrane Proteins from Mouse Brain Tissue

Source: Speca, D. J., et al. Acyl-PEGyl Exchange Gel Shift Assay for Quantitative Determination of Palmitoylation of Brain Membrane Proteins. J. Vis. Exp. ...

Take cell membrane proteins from mouse brain tissue.
These proteins exhibit post-translational modification with palmitoyl groups, a fatty acid, attached to cysteine residues.
Add a reducing buffer and incubate at a high temperature to break bonds between non-palmitoylated cysteines, exposing them.
Incubate with a blocking reagent that binds to the exposed cysteines, leaving only the palmitoylated cysteines reactive.
Transfer the solution, mix it with chloroform, methanol, and deionized water, centrifuge it to precipitate the proteins, and remove any impurities. Next, add methanol, centrifuge, and discard any remaining contaminants.
Add a buffer, sonicate to solubilize the pellet, then centrifuge and remove insoluble materials.
Apply a cleaving reagent to remove the palmitoyl groups from the palmitoylated cysteines.
Repeat chloroform-methanol precipitation and remove any impurities.
Incubate with a polymer that binds to unblocked cysteines, helping to identify the palmitoylated proteins.
Repeat chloroform-methanol precipitation and remove any unbound polymers.
Resuspend the pellet for further analysis of the palmitoylation state of the isolated proteins.

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45 Görüntüleme. Source: Speca, D. J., et al. Acyl-PEGyl Exchange Gel Shift Assay for Quantitative Determination of Palmitoylation of Brain Membrane Proteins. J. Vis. Exp. (2020). The video demonstrates the analysis of the palmitoylation state of cell membrane proteins isolated from mouse brain tissue. The process involves exposing the non-palmitoylated cysteines and blocking them, then using a polymer that binds to the palmitoylated cysteines, aiding in assessing the palmitoylation state of the isolated protei...

Video: Assessing the Palmitoylation State of Cell Membrane Proteins from Mouse Brain Tissue - Kavram

Extraction of Extracellular Vesicles from Whole Tissue

Circulating and interstitial small membrane-bound extracellular vesicles (EVs) represent promising targets for the development of novel diagnostic or prognostic biomarker assays, and likely serve as important players in the progression of a vast spectrum of diseases. Current research is focused on the characterization of vesicles secreted from multiple cell and tissue types in order to better understand the role of EVs in the pathogenesis of conditions including neurodegeneration, inflammation, and cancer. However, globally consistent and reproducible techniques to isolate and purify vesicles remain in progress. Moreover, methods for extraction of EVs from solid tissue ex vivo are scarcely described. Here, we provide a detailed protocol for extracting small EVs of interest from whole fresh or frozen tissues, including brain and tumor specimens, for further characterization. We demonstrate the adaptability of this method for multiple downstream analyses, including electron microscopy and immunophenotypic characterization of vesicles, as well as quantitative mass spectrometry of EV proteins.

Here, we provide a detailed protocol to isolate small extracellular vesicles (EVs) from whole tissues, including brain and tumor specimens. This method offers a reproducible technique to extract EVs from solid tissue for further downstream analyses.

Hücre dışı veziküller çekme--dan tüm doku

Circulating and interstitial small membrane-bound extracellular vesicles (EVs) represent promising targets for the development of novel diagnostic or ...

JoVE Journal

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Detection of Protein S-Acylation using Acyl-Resin Assisted Capture

Protein S-acylation, also referred to as S-palmitoylation, is a reversible post-translational modification of cysteine residues with long-chain fatty acids via a labile thioester bond. S-acylation, which is emerging as a widespread regulatory mechanism, can modulate almost all aspects of the biological activity of proteins, from complex formation to protein trafficking and protein stability. The recent progress in understanding of the biological function of protein S-acylation was achieved largely due to the development of novel biochemical tools allowing robust and sensitive detection of protein S-acylation in a variety of biological samples. Here, we describe acyl resin-assisted capture (Acyl-RAC), a recently developed method based on selective capture of endogenously S-acylated proteins by thiol-reactive Sepharose beads. Compared to existing approaches, Acyl-RAC requires fewer steps and can yield more reliable results when coupled with mass spectrometry for identification of novel S-acylation targets. A major limitation in this technique is the lack of ability to discriminate between fatty acid species attached to cysteines via the same thioester bond.

Acyl-RAC (Acyl-Resin Assisted Capture) is a highly sensitive, reliable and easy to perform method to detect reversible lipid modification of cysteine residues (S-acylation) in a variety of biological samples.

Acyl-Reşin Destekli Yakalama ile Protein S-Acylation Tespiti

Protein S-acylation, also referred to as S-palmitoylation, is a reversible post-translational modification of cysteine residues with long-chain fatty ...

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Determination of Plasma Membrane Partitioning for Peripherally-associated Proteins

This method provides a fast approach for the determination of plasma membrane partitioning of any fluorescently-tagged peripherally-associated protein using the profiles of fluorescence intensity across the plasma membrane. Measured fluorescence profiles are fitted by a model for membrane and cytoplasm fluorescence distribution along a line applied perpendicularly to the cell periphery. This model is constructed from the fluorescence intensity values in reference cells expressing a fluorescently-tagged marker for cytoplasm and with FM 4-64-labeled plasma membrane. The method can be applied to various cell types and organisms; however, only plasma membranes of non-neighboring cells can be evaluated. This fast microscopy-based method is suitable for experiments, where subtle and dynamic changes of plasma membrane-associated markers are expected and need to be quantified, e.g., in the analysis of mutant versions of proteins, inhibitor treatments, and signal transduction observations. The method is implemented in a multi-platform R package that is coupled with an ImageJ macro that serves as a user-friendly interface.

Here, we present a protocol to perform a quantitative analysis of the level of plasma-membrane association for fluorescently-tagged peripherally-associated protein. The method is based on the computational decomposition of membrane and cytoplasmic component of signal observed in cells labeled with plasma membrane fluorescent marker.

Plazma zarı periferik ilişkili proteinler için bölümleme belirlenmesi

This method provides a fast approach for the determination of plasma membrane partitioning of any fluorescently-tagged peripherally-associated protein ...

Assessing the Palmitoylation State of Cell Membrane Proteins from Mouse Brain Tissue

TRANSKRIPT

Assessing the Palmitoylation State of Cell Membrane Proteins from Mouse Brain Tissue

Hücre dışı veziküller çekme--dan tüm doku

Acyl-Reşin Destekli Yakalama ile Protein S-Acylation Tespiti

Plazma zarı periferik ilişkili proteinler için bölümleme belirlenmesi