The authors thank K. Woolfrey for advice and input on the APEGS assay. These studies were funded by research grants to E.D. from the Whitehall Foundation and the NIH-NIMH (1R01MH119347).

All animal procedures followed guidelines set forth by the National Institutes of Health (NIH) and have been approved by the Institutional Animal Care and Use Committee at the University of California, Davis.
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 g 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 g for 10 min at 4 &#176;C.</li>
	<li>Combine the supernatants from step 1.2 and centrifuge at 40,000 x g 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 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 g 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; 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; 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 dH2O and vortex briefly.</li>
		<li>Centrifuge the samples at 3,000 x g 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 g 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 protein precipitate in 125 &#181;L of buffer A and sonicate briefly. Centrifuge at &#62;13,000 x g 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+; Table 1) or buffer T (HAM-; 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; 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 g 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 g 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 g 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). Reserve 5 &#181;L for BCA protein quantitation. After quantitation, add an appropriate amount of 4x Laemmli sample buffer and, depending on the protein of interest, potentially heat the sample to 70&#8722;100 &#176;C for 10 min. 
		NOTE: Boiling may cause aggregation of some membrane proteins.</li>
	</ol>
	</li>
</ol>
3. Western blot analysis of PEGylated proteins
<ol>
	<li>Load the sample onto an sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel13. 
	NOTE: In this protocol 10% polyacrylamide was used.</li>
	<li>Use standard separation, transfer and detection protocols for Western blotting14.</li>
	<li>Use densitometry14 to determine the relative amounts of PEGylated versus nonPEGylated proteins.</li>
</ol>

<ol>
	<li>Blaskovic, S., Blanc, M., van der Goot, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+does+S-palmitoylation+do+to+membrane+proteins?.">What does S-palmitoylation do to membrane proteins?.</a> FEBS Journal. 280, 2766-2774 (2013).</li><li>Fukata, Y., Fukata, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+palmitoylation+in+neuronal+development+and+synaptic+plasticity.">Protein palmitoylation in neuronal development and synaptic plasticity.</a> Nature Reviews Neuroscience. 11, 161-175 (2010).</li><li>Globa, A. K., Bamji, S. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+palmitoylation+in+the+development+and+plasticity+of+neuronal+connections.">Protein palmitoylation in the development and plasticity of neuronal connections.</a> Current Opinion in Neurobiology. 45, 210-220 (2017).</li><li>Thomas, G. M., Huganir, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Palmitoylation-dependent+regulation+of+glutamate+receptors+and+their+PDZ+domain-containing+partners.">Palmitoylation-dependent regulation of glutamate receptors and their PDZ domain-containing partners.</a> Biochemical Society Transactions. 41, 72-78 (2013).</li><li>Kalashnikova, E., 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=SynDIG1:+an+activity-regulated,+AMPA-+receptor-interacting+transmembrane+protein+that+regulates+excitatory+synapse+development.">SynDIG1: an activity-regulated, AMPA- receptor-interacting transmembrane protein that regulates excitatory synapse development.</a> Neuron. 65, 80-93 (2010).</li><li>Kaur, 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=Activity-Dependent+Palmitoylation+Controls+SynDIG1+Stability,+Localization,+and+Function.">Activity-Dependent Palmitoylation Controls SynDIG1 Stability, Localization, and Function.</a> Journal of Neuroscience. 36, 7562-7568 (2016).</li><li>Wan, J., Roth, A. F., Bailey, A. O., Davis, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Palmitoylated+proteins:+purification+and+identification.">Palmitoylated proteins: purification and identification.</a> Nature Protocols. 2, 1573-1584 (2007).</li><li>Howie, 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=Substrate+recognition+by+the+cell+surface+palmitoyl+transferase+DHHC5.">Substrate recognition by the cell surface palmitoyl transferase DHHC5.</a> Proceedings of the National Academy of Sciences of the United States of America. 111, 17534-17539 (2014).</li><li>Kanadome, T., Yokoi, N., Fukata, Y., Fukata, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+Screening+of+Depalmitoylating+Enzymes+and+Evaluation+of+Their+Activities+by+the+Acyl-PEGyl+Exchange+Gel-Shift+(APEGS)+Assay.">Systematic Screening of Depalmitoylating Enzymes and Evaluation of Their Activities by the Acyl-PEGyl Exchange Gel-Shift (APEGS) Assay.</a> Methods in Molecular Biology. 2009, 83-98 (2019).</li><li>Percher, 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=Mass-tag+labeling+reveals+site-specific+and+endogenous+levels+of+protein+S-fatty+acylation.">Mass-tag labeling reveals site-specific and endogenous levels of protein S-fatty acylation.</a> Proceedings of the National Academy of Sciences of the United States of America. 113, 4302-4307 (2016).</li><li>Percher, A., Thinon, E., Hang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass-Tag+Labeling+Using+Acyl-PEG+Exchange+for+the+Determination+of+Endogenous+Protein+S-Fatty+Acylation.">Mass-Tag Labeling Using Acyl-PEG Exchange for the Determination of Endogenous Protein S-Fatty Acylation.</a> Current Protocols in Protein Science. 89, 14.17.1-14.17.11 (2017).</li><li>Yokoi, N., 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+PSD-95+Depalmitoylating+Enzymes.">Identification of PSD-95 Depalmitoylating Enzymes.</a> Journal of Neuroscience. 36, 6431-6444 (2016).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=JoVE+Science+Education+Database.+Separating+Protein+with+SDS-PAGE.">JoVE Science Education Database. Separating Protein with SDS-PAGE.</a> Basic Methods in Cellular and Molecular Biology. , (2019).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Science+Education+Database.+The+Western+Blot.">Science Education Database. The Western Blot.</a> Basic Methods in Cellular and Molecular Biology. , (2019).</li><li>Chenaux, 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=Loss+of+SynDIG1+Reduces+Excitatory+Synapse+Maturation+But+Not+Formation+In+Vivo.">Loss of SynDIG1 Reduces Excitatory Synapse Maturation But Not Formation In Vivo.</a> eNeuro. 3 (5), (2016).</li><li>Matt, 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=SynDIG4/Prrt1+Is+Required+for+Excitatory+Synapse+Development+and+Plasticity+Underlying+Cognitive+Function.">SynDIG4/Prrt1 Is Required for Excitatory Synapse Development and Plasticity Underlying Cognitive Function.</a> Cell Reports. 22, 2246-2253 (2018).</li><li>Purkey, A. 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=AKAP150+Palmitoylation+Regulates+Synaptic+Incorporation+of+Ca(2+)-Permeable+AMPA+Receptors+to+Control+LTP.">AKAP150 Palmitoylation Regulates Synaptic Incorporation of Ca(2+)-Permeable AMPA Receptors to Control LTP.</a> Cell Reports. 25, 974-987 (2018).</li><li>Woolfrey, K. M., Sanderson, J. L., Dell'Acqua, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+palmitoyl+acyltransferase+DHHC2+regulates+recycling+endosome+exocytosis+and+synaptic+potentiation+through+palmitoylation+of+AKAP79/150.">The palmitoyl acyltransferase DHHC2 regulates recycling endosome exocytosis and synaptic potentiation through palmitoylation of AKAP79/150.</a> Journal of Neuroscience. 35, 442-456 (2015).</li></ol>

The authors have nothing to disclose.

In our previous work, we utilized the ABE assay to demonstrate that SynDIG1 is palmitoylated at two conserved juxta-transmembrane Cys residues (found in all SynDIG proteins) in an activity-dependent manner to regulate stability, localization, and function6. A limitation is that the ABE assay requires affinity purification with agarose resins conjugated to avidin moieties as the final step in the procedure, resulting in significant loss of signal that complicate quantitative analysis. Furthermore, ...

S-palmitoylation is a reversible post-translational modification of target proteins that regulates stable membrane association, protein trafficking, and protein-protein interactions1. It involves addition of a 16-carbon palmitate moiety to cysteine residues via thioester linkage catalyzed by palmitoyl acyltransferase (PAT) enzymes. Many synaptic proteins in the brain are palmitoylated, including AMPA-Rs and PSD-95, in an activity-dependent manner to regulate stability, localization, and function2,3,4. Alterations in the levels of synaptic AMPARs in the PSD via interaction of auxiliary factors with synaptic scaffolds such as PSD-95 underlies synaptic plasticity; thus, methods to determine the palmitoylation state of synaptic proteins provides important insight into mechanisms of synaptic plasticity.
Previously, we identified the SynDIG family of four genes (SynDIG1-4) encoding brain-specific transmembrane proteins that associate with AMPARs5. Overexpression or knock-down of SynDIG1 in dissociated rat hippocampal neurons increases or decreases, respectively, AMPA-R synapse size and number by ~50% as detected using immunocytochemistry and electrophysiology5. We utilized the acyl-biotin exchange (ABE) assay to demonstrate that SynDIG1 is palmitoylated at two conserved juxta-transmembrane Cys residues (found in all SynDIG proteins) in an activity-dependent manner to regulate stability, localization, and function6. The ABE assay relies on exchange of biotin on cysteines protected by modification and subsequent affinity purification7. Here, we describe an innovative biochemical approach, the acyl-PEGyl exchange gel shift (APEGS) assay8,9,10,11,12, which does not require affinity purification and instead utilizes changes in gel mobility to determine the number of modifications for a protein of interest. The protocol is described for investigation of endogenous membrane proteins from mouse brain for which suitable antibodies are available.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Hydroxylamine (HAM)</td>
			<td>ThermoFisher</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) phosphine-hydrochloride (TCEP)</td>
			<td>EMD Millipore</td>
			<td>580560</td>
			<td></td>
		</tr>
	</tbody>
</table>

acyl-pegyl exchange gel shift assay for quantitative determination of palmitoylation of brain membrane proteins

Activity-dependent alterations in the levels of synaptic AMPA receptors (AMPARs) within the postsynaptic density (PSD) is thought to represent a cellular mechanism for learning and memory. Palmitoylation regulates localization and function of many synaptic proteins including AMPA-Rs, auxiliary factors and synaptic scaffolds in an activity-dependent manner. We identified the synapse differentiation induced gene (SynDIG) family of four genes (SynDIG1-4) encoding brain-specific transmembrane proteins that associate with AMPARs and regulate synapse strength. SynDIG1 is palmitoylated at two cysteine residues located at positions 191 and 192 in the juxta-transmembrane region important for activity-dependent excitatory synapse development. Here, we describe an innovative biochemical approach, the acyl-PEGyl exchange gel shift (APEGS) assay, to investigate the palmitoylation state of any protein of interest and demonstrate its utility with the SynDIG family of proteins in mouse brain lysates.

Immunoblotting with antibodies against the protein of interest reveals the palmitoylated state (non, singly, doubly, etc.) in mouse brain lysates as determined by mobility shift compared with samples in which HAM was not included. Previously, we had demonstrated that SynDIG1 was palmitoylated at two sites using the ABE assay6; however, we could not determine whether both sites were modified in brain tissue. Here we show that SynDIG1, SynDIG4/Prrt1, and Prrt2 are pa...

Watch this Scientific Journal Video about Acyl-PEGyl Exchange Gel Shift Assay for Quantitative Determination of Palmitoylation of Brain Membrane Proteins at JoVE.com

Acyl-PEGyl Exchange Gel Shift Assay for Quantitative Determination of Palmitoylation of Brain Membrane Proteins

Palmitoylation entails the incorporation of a 16-carbon palmitate moiety to cysteine residues of target proteins in a reversible manner. Here, we describe a biochemical approach, the acyl-PEGyl exchange gel shift (APEGS) assay, to investigate the palmitoylation state of any protein of interest in mouse brain lysates.

Activity-dependent alterations in the levels of synaptic AMPA receptors (AMPARs) within the postsynaptic density (PSD) is thought to represent a cellular ...

acyl-pegyl-exchange-gel-shift-assay-for-quantitative-determination

Research

JoVE Journal

Biochemistry

6.5K Views.  UC Davis School of Medicine. Palmitoylation entails the incorporation of a 16-carbon palmitate moiety to cysteine residues of target proteins in a reversible manner. Here, we describe a biochemical approach, the acyl-PEGyl exchange gel shift (APEGS) assay, to investigate the palmitoylation state of any protein of interest in mouse brain lysates.

Video: Acyl-PEGyl Exchange Gel Shift Assay for Quantitative Determination of Palmitoylation of Brain Membrane Proteins

An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. Radioactivity has been the predominant method of DNA labeling in EMSAs. However, recent advances in fluorescent dyes and scanning methods have prompted the use of fluorescent tagging of DNA as an alternative to radioactivity for the advantages of easy handling, saving time, reducing cost, and improving safety. We have recently used fluorescent EMSA (fEMSA) to successfully address an important biological question. Our fEMSA analysis provides mechanistic insight into the effect of a missense mutation, G73E, in the highly conserved HMG transcription factor SOX-2 on olfactory neuron type diversification. We found that mutant SOX-2G73E protein alters specific DNA binding activity, thereby causing olfactory neuron identity transformation. Here, we present an optimized and cost-effective step-by-step protocol for fEMSA using infrared fluorescent dye-labeled oligonucleotides containing the LIM-4/SOX-2 adjacent target sites and purified SOX-2 proteins (WT and mutant SOX-2G73E proteins) as a biological example.

We describe here an optimized protocol of fluorescent Electrophoretic Mobility Shift Assays (fEMSA) using purified SOX-2 proteins together with infrared fluorescent dye-labeled DNA probes as a case study to tackle an important biological question.

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. ...

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule level in multiparameter fluorescence detection (MFD) mode is presented here. MFD maximizes the usage of all &#34;dimensions&#34; of fluorescence to reduce photophysical and experimental artifacts and allows for the measurement of interdye distance with an accuracy up to ~1 &#197; in rigid biomolecules. This method was used to identify three conformational states of the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor to explain the activation of the receptor upon ligand binding. When comparing the known crystallographic structures with experimental measurements, they agreed within less than 3 &#197; for more dynamic biomolecules. Gathering a set of distance restraints that covers the entire dimensionality of the biomolecules would make it possible to provide a structural model of dynamic biomolecules.

A protocol for high-precision FRET experiments at the single molecule level is presented here. Additionally, this methodology can be used to identify three conformational states in the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor. Determining precise distances is the first step towards building structural models based on FRET experiments.

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule ...

Oligopeptide Competition Assay for Phosphorylation Site Determination

Protein phosphorylation at specific sites determines its conformation and interaction with other molecules. Thus, protein phosphorylation affects biological functions and characteristics of the cell. Currently, the most common method for discovering phosphorylation sites is by liquid chromatography/mass spectrometry (LC/MS) analysis, a rapid and sensitive method. However, relatively labile phosphate moieties are often released from phosphopeptides during the fragmentation step, which often yields false-negative signals. In such cases, a traditional in vitro kinase assay using site-directed mutants would be more accurate, but this method is laborious and time-consuming. Therefore, an alternative method using peptide competition may be advantageous. The consensus recognition motif of 5&#39; adenosine monophosphate-activated protein kinase (AMPK) has been established1 and was validated using a positional scanning peptide library assay2. Thus, AMPK phosphorylation sites for a novel substrate could be predicted and confirmed by the peptide competition assays. In this report, we describe the detailed steps and procedures for the in vitro oligopeptide-competing kinase assay by illustrating AMPK-mediated nuclear factor erythroid 2-related factor 2 (Nrf2) phosphorylation. To authenticate the phosphorylation site, we carried out a sequential in vitro kinase assay using a site-specific mutant. Overall, the peptide competition assay provides a method to screen multiple potential phosphorylation sites and to identify sites for validation by the phosphorylation site mutants.

Peptide competition assays are widely used in a variety of molecular and immunological experiments. This paper describes a detailed method for an in vitro oligopeptide-competing kinase assay and the associated validation procedures, which may be useful to find specific phosphorylation sites.

Protein phosphorylation at specific sites determines its conformation and interaction with other molecules. Thus, protein phosphorylation affects ...

Detection of Detergent-sensitive Interactions Between Membrane Proteins

Our ability to explore protein-protein interactions is the key to understanding regulatory connections in the cell. However, detection of protein-protein interactions in many cases is associated with significant experimental challenges. In particular, sorting receptors interact with their protein cargo in the lumen of the membrane compartments often in a detergent-sensitive fashion, making co-immunoprecipitation of these proteins unusable. Binding of the sorting receptor sortilin to glucose transporter GLUT4 may serve as an example of weak luminal interactions between membrane proteins. Here, we describe a fast, simple, and inexpensive assay to validate the interaction between sortilin and GLUT4. For that, we have designed and chemically synthesized the myc-tagged peptide corresponding to the potential sortilin-binding epitope in the luminal part of GLUT4. Sortilin tagged with six histidines was expressed in mammalian cells, and isolated from cell lysates using Cobalt beads. Sortilin immobilized on the beads was incubated with the peptide solution at different pH values, and the eluted material was analyzed by Western blotting. This assay can be easily adapted to study other detergent-sensitive protein-protein interactions.

We describe a protocol for detection of detergent-sensitive interactions between membrane proteins using binding of the sorting receptor, sortilin, to the first luminal loop of the glucose transporter protein, GLUT4, as an example.

Our ability to explore protein-protein interactions is the key to understanding regulatory connections in the cell. However, detection of protein-protein ...

Analysis of Thylakoid Membrane Protein Complexes by Blue Native Gel Electrophoresis

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

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

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

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

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

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

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

Use of Recombinant Fusion Proteins in a Fluorescent Protease Assay Platform and Their In-gel Renaturation

Proteases are intensively studied enzymes due to their essential roles in several biological pathways of living organisms and in pathogenesis; therefore, they are important drug targets. We have developed a magnetic-agarose-bead-based assay platform for the investigation of proteolytic activity, which is based on the use of recombinant fusion protein substrates. In order to demonstrate the use of this assay system, a protocol is presented on the example of human immunodeficiency virus type 1 (HIV-1) protease. The introduced assay platform can be utilized efficiently in the biochemical characterization of proteases, including enzyme activity measurements in mutagenesis, kinetic, inhibition, or specificity studies, and it may be suitable for high-throughput substrate screening or may be adapted to other proteolytic enzymes.
In this assay system, the applied substrates contain N-terminal hexahistidine (His6) and maltose-binding protein (MBP) tags, cleavage sites for tobacco etch virus (TEV) and HIV-1 proteases, and a C-terminal fluorescent protein. The substrates can be efficiently produced in Escherichia coli cells and easily purified using nickel (Ni)-chelate-coated beads. During the assay, the proteolytic cleavage of bead-attached substrates leads to the release of fluorescent cleavage fragments, which can be measured by fluorimetry. Additionally, cleavage reactions can be analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A protocol for the in-gel renaturation of assay components is also described, as partial renaturation of fluorescent proteins enables their detection based on molecular weight and fluorescence.

Here, we present the detailed procedure of a recently developed protease assay platform utilizing N-terminal hexahistidine/maltose-binding protein and fluorescent protein-fused recombinant substrates attached to the surface of nickel-nitrilotriacetic acid magnetic agarose beads. A subsequent in-gel analysis of the assay samples separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is also presented.

Proteases are intensively studied enzymes due to their essential roles in several biological pathways of living organisms and in pathogenesis; therefore, ...

Robust Comparison of Protein Levels Across Tissues and Throughout Development Using Standardized Quantitative Western Blotting

Western blotting is a technique that is commonly used to detect and quantify protein expression. Over the years, this technique has led to many advances in both basic and clinical research. However, as with many similar experimental techniques, the outcome of Western blot analyses is easily influenced by choices made in the design and execution of the experiment. Specific housekeeping proteins have traditionally been used to normalize protein levels for quantification, however, these have a number of limitations and have therefore been increasingly criticized over the past few years. Here, we describe a detailed protocol that we have developed to allow us to undertake complex comparisons of protein expression variation across different tissues, mouse models (including disease models), and developmental timepoints. By using a fluorescent total protein stain and introducing the use of an internal loading standard, it is possible to overcome existing limitations in the number of samples that can be compared within experiments and systematically compare protein levels across a range of experimental conditions. This approach expands the use of traditional western blot techniques, thereby allowing researchers to better explore protein expression across different tissues and samples.

This method describes a robust and reproducible approach for the comparison of protein levels in different tissues and at different developmental timepoints using a standardized quantitative western blotting approach.

Western blotting is a technique that is commonly used to detect and quantify protein expression. Over the years, this technique has led to many advances ...

Interactions with and Membrane Permeabilization of Brain Mitochondria by Amyloid Fibrils

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary mechanism of amyloid aggregate-induced toxicity in neurodegenerative diseases. However, most reports describing the mechanisms of membrane disruption are based on phospholipid model systems, and studies directly targeting events occurring at the level of biological membranes are rare. Described here is a model for studying the mechanisms of amyloid toxicity at the membrane level. For mitochondrial isolation, density gradient medium is used to obtain preparations with minimal myelin contamination. After mitochondrial membrane integrity confirmation, the interaction of amyloid fibrils arising from &#945;-synuclein, bovine insulin, and hen egg white lysozyme (HEWL) with rat brain mitochondria, as an in vitro biological model, is investigated. The results demonstrate that treatment of brain mitochondria with fibrillar assemblies can cause different degrees of membrane permeabilization and ROS content enhancement. This indicates structure-dependent interactions between amyloid fibrils and mitochondrial membrane. It is suggested that biophysical properties of amyloid fibrils and their specific binding to mitochondrial membranes may provide explanations for some of these observations.

Provided here is a protocol for investigating the interactions between native form, prefibrillar, and mature amyloid fibrils of different peptides and proteins with mitochondria isolated from different tissues and various areas of the brain.

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary ...

Transverse Sectioning of Mature Rice (Oryza sativa L.) Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm phenotyping can be used to classify genotypes based on SG morphotype using scanning electron microscopic (SEM) analysis. SGs can be visualized using SEM by slicing through the kernel (pericarp, aleurone layers, and endosperm) and exposing the organellar contents. Current methods require the rice kernel to be embedded in plastic resin and sectioned using a microtome or embedded in a truncated pipette tip and sectioned by hand using a razor blade. The former method requires specialized equipment and is time-consuming, while the latter introduces a new host of problems depending on rice genotype. Chalky rice varieties, particularly, pose a problem for this type of sectioning due to the friable nature of their endosperm tissue. Presented here is a technique for preparing translucent and chalky rice kernel sections for microscopy, requiring only pipette tips and a scalpel blade. Preparing the sections within the confines of a pipette tip prevents rice kernel endosperm from shattering (for translucent or &#8216;vitreous&#8217; phenotypes) and crumbling (for chalky phenotypes). Using this technique, endosperm cell patterning and the structure of intact SGs can be observed.

Transverse Sectioning of Mature Rice Oryza sativa L. Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

This protocol allows for the preparation of transverse sections of cereal seeds (e.g., rice) for the analysis of endosperm and starch granule morphology using scanning electron microscopy.