Name: assessing the palmitoylation state of cell membrane proteins from mouse brain tissue
Uploaded: 2025-03-31T15:00:42.000+00:00
Duration: 1 min 30 s
Description: Source: Speca, D. J., et al. Acyl-PEGyl Exchange Gel Shift Assay for Quantitative Determination of Palmitoylation of Brain Membrane Proteins. J. Vis. Exp. ...

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

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

First, disrupt the disulfide bonds by incubating the solubilized protein in 25 microliters of TCEP, for 60 minutes at 55 degrees Celsius. Then, block free cysteines by incubating the solution with 12.5 microliters of the stock solution of NEM for three hours at room temperature.
Next, begin the process of chloroform-methanol precipitation. Transfer the protein solution from the 1.5-milliliter tube to a polypropylene or glass tube that can be centrifuged in a swinging bucket rotor at modest speed. Add 2 milliliters of methanol, and vortex briefly. Add 1 milliliter of chloroform, and vortex briefly. Then, add 1.5 milliliters of deionized water and vortex briefly again.
If necessary, invert the tube to ensure thorough mixing. Centrifuge the samples at 3,000 times G and 25 degrees Celsius for 30 minutes in a swinging bucket rotor. Carefully remove and discard the upper phase of the solution in a tube. Add 1.5 milliliters of methanol. Mix gently but thoroughly by gentle inversion of the tube, being careful not to fragment the opaque protein pancake.
Centrifuge the sample at 3,000 times G and 25 degrees Celsius for 10 minutes in a swinging bucket rotor. Using a glass serological pipette, remove as much of the top phase of the solution as possible without disturbing the protein pancake. Carefully rinse the pellet with 1 milliliter of methanol, and allow it to air dry for at least 10 minutes.
With the chloroform-methanol precipitation complete, begin the cleavage of the palmitoyl thioester linkages by resuspending the protein precipitate in 125 microliters of buffer A. Transfer to a new 1.5-milliliter tube. Sonicate the tube briefly, and then centrifuged it at 13,000 times G or higher and 25 degrees Celsius for 10 minutes to remove insoluble material.
Transfer the solubilized protein to new 1.5-milliliter tubes, and add 375 microliters of buffer H or buffer T. After incubating the samples for 60 minutes at room temperature, repeat the chloroform-methanol precipitation. To add mPEG to unprotected cysteines, begin by resuspending the pellet in 100 microliters of buffer A containing 10 millimolar TCEP.
Then transfer the suspension to a 1.5 milliliter tube. Add 25 microliters of stock mPEG 5k solution and mix by pipetting. Incubate at room temperature with end over end rotation. After 60 minutes of incubation, remove unincorporated mPEG 5k by performing chloroform methanol precipitation again.
Centrifuge at greater than 13,000 times G and 25 degrees Celsius for 10 minutes. Carefully remove the upper phase as before, avoiding the thick flocculent pancake. Add 1 milliliter of methanol and mix gently but thoroughly. Centrifuge at greater than 13,000 times G and 25 degrees Celsius for 10 minutes.
Carefully remove the supernatant and rinse the pellet with 1 milliliter of methanol. Again, centrifuge at greater than 13,000 times G and 25 degrees Celsius for 10 minutes. After air drying the pellet, resuspended in 50 microliters of buffer A without TCEP or any reducing agent.

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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>

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>

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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视频: Assessing the Palmitoylation State of Cell Membrane Proteins from Mouse Brain Tissue - 实验

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

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