Name: A Technique for Stabilizing Membrane Proteins in Nanodiscs (Video) | JoVE Journal
Uploaded: 2026-04-30T13:20:00.000+00:00
Duration: 10 min 21 s
Description: Learn a robust protocol for reconstituting the human potassium channel Task2 into nanodiscs using membrane scaffold proteins and phospholipids. This method enables high-resolution cryo-EM structural analysis.

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5 minutes

The authors thank the staff of the cryo-EM Center at Henan University, as well as Professor Wei Wang, for their expert support. Funding was provided by the National Natural Science Foundation of China (Grants 32371261 to B.L. and 32301011 to R.Z.).

1. Preparation of lipid stock
<ol>
	<li>Lipid Preparation
	<ol>
		<li>Prepare lipid mixtures using three phospholipids: 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 2-oleoyl-1-palmitoylglycero-3-phosphocholine (POPC), and 1-palmitoyl-2-oleoylphosphatidylserine (POPS).</li>
		<li>Use pre-prepared stock solutions of these lipids in chloroform, each at a concentration of 25 mg/mL. 
		NOTE: Personal protective equipment is required when handling chloroform, and all procedures must be conducted in a fume hood. Store chloroform in a dedicated chemical cabinet.</li>
		<li>Due to the volatility of chloroform, when measuring with a microsyringe, verify that the volume of the lipid stock solution matches the initially recorded volume before each use. If evaporation occurs, replenish the solution with pure chloroform to the original volume and mix thoroughly. 
		NOTE: Before each measurement, rinse the microsyringe twice with chloroform.</li>
		<li>Prepare a 100 &#181;L lipid mixture with a mass ratio of DOPE:POPC:POPS = 2:1:1. Based on this ratio, combine 50 &#181;L of VDOPE, 25 &#181;L of VPOPC, and 25 &#181;L of VPOPS. Measure the corresponding volume according to the calculated number. Add it to the glass test tube and mix thoroughly.</li>
		<li>Place the glass test tube in a water bath to reduce the volatilization of chloroform and keep the ambient temperature stable.</li>
	</ol>
	</li>
	<li>Lipid film preparation
	<ol>
		<li>Evaporate the chloroform under a gentle stream of nitrogen gas until all liquid has been removed and a transparent, homogeneous gel-like film forms at the bottom of the test tube. 
		NOTE: Ensure a uniform, dry lipid film is visible at the bottom of the glass tube, with no residual liquid droplets. Avoid excessive nitrogen flow, as it may cause bubbling and prevent complete drying of the chloroform.)</li>
		<li>Add approximately 2 mL of n-pentane dropwise to a glass test tube. Then evaporate the solvent using the nitrogen-drying method described in 1.2.1 to obtain a dry, transparent lipid film. n-Pentane is evaporated in a glass test tube due to its chemical inertness and compatibility with organic solvents. Glass avoids the risk of plastic leaching or degradation associated with many plastic labware materials, thereby preserving lipid purity during film formation. 
		NOTE: n-Pentane must be stored in a dedicated reagent cabinet and, when used, handled with care in a fume hood while wearing appropriate personal protective equipment.</li>
		<li>Loosen the cap of the test tube and place it in a vacuum desiccator. Evacuate the desiccator and maintain under vacuum to remove any residual solvents. Place self-indicating silica gel at the bottom of the desiccator to absorb moisture. 
		NOTE: Evaporate residual chloroform and n-pentane to dryness in a fume hood.</li>
	</ol>
	</li>
	<li>Lipid hydration and stock solution preparation
	<ol>
		<li>Remove the lipid film from the vacuum&#160;desiccator and hydrate it by adding 250 &#181;L of detergent-free low salt buffer (LSB) (Supplementary Table 1). This yields a final lipid concentration of 10 mg/mL. 
		NOTE: A potassium ion-based buffer was selected because Task2 is a potassium ion channel</li>
		<li>After resuspending the lipid, divide the lipid evenly into four glass tubes and sonicate the suspensions until they become clear and transparent, indicating successful liposome formation (approximately 1.5 h). Perform sonication at 60% amplitude using a cycle of 4 s on and 6 s off.</li>
		<li>Pool the clarified lipid solution and aliquot 1.5 mL into individual microcentrifuge tubes. Immediately flush the headspace of each tube with nitrogen gas and store at -80 &#176;C until use.</li>
	</ol>
	</li>
</ol>
2. Over-expression and purification of MSP
<ol>
	<li>Prokaryotic expression of MSP
	<ol>
		<li>Plasmid transformation: Use the MSP1D1 plasmid carrying a 7&#215;His tag to transform the E. coli BL21 (DE3) competent cells. Plate the transformation mixture onto Luria-Bertani (LB) agar plates containing kanamycin (50 &#181;g/mL) and incubate overnight at 37 &#176;C to obtain single colonies.</li>
		<li>Culture amplification: Pick a single clone and inoculate it into 5 mL of LB medium containing kanamycin and incubate overnight at 37 &#176;C with shaking. Inoculate the overnight culture into liquid LB medium at a 1:100 ratio, then grow at 37 &#176;C with shaking until the optical density at 600 nm (OD600) reaches 2.0&#8211;3.0.</li>
		<li>Protein expression induction: Induce MSP expression by adding isopropyl &#946;-d-1-thiogalactopyranoside (IPTG) to a final concentration of 0.5&#8211;1 mM. Continue culturing the cells at 16 &#176;C for 16&#8211;20 h with shaking.</li>
	</ol>
	</li>
	<li>Cell disruption
	<ol>
		<li>Pellet Collection: Centrifuge the bacterial culture at 4 &#176;C for 10 min. Discard the supernatant and retain the cell pellet.</li>
		<li>Bacteria lysis: Resuspend the pellet derived from 1 L of culture in 100&#8211;150 mL of Lysis buffer (Supplementary Table 1). Homogenize thoroughly until a uniform suspension with no visible clumps is achieved. Add PMSF to the suspension to inhibit proteases and reduce foaming.</li>
		<li>Sonication: Use a refrigerated ultrasonic cell disruptor to break the bacteria. Set the power to 35 % and apply cycles of 3 s pulses followed by 6 s pauses. To prevent overheating, allow a 2 min rest interval after every 15 min of cumulative sonication time. Continue until the lysate becomes clear and light brown in color.</li>
		<li>Clarification: Centrifuge the lysate at 30,000 g for 45 min at 4 &#176;C and collect the supernatant for subsequent steps.</li>
	</ol>
	</li>
	<li>MSP purification
	<ol>
		<li>Ni-NTA Resin Preparation: Take 3&#8211;5 mL of Ni-NTA resin and pack it into a gravity column (size: 26.4&#215;134.8 mm). Wash the resin with 3 column volumes (CV) of distilled water and equilibrate it with 3 CV of Wash Buffer 1 (Supplementary Table 1).</li>
		<li>Binding of MSP to the Resin: Following centrifugation of the cell lysate, mix the supernatant with the equilibrated Ni-NTA resin. Add imidazole to a final concentration of 5 mM and incubate the mixture at 4 &#176;C for 2 h with gentle rotation in the dark.</li>
		<li>Wash to remove impurities: Sequentially wash with gradients of Wash Buffer 1, Wash Buffer 2 (Supplementary Table 1), and Wash Buffer 3 (Supplementary Table 1), using at least 50 mL of each buffer. Monitor the UV absorbance at 280 nm (A280) of the flow-through and continue washing until the A280 value falls below 0.01.</li>
		<li>Elution: Elute the target protein with Elution Buffer (Supplementary Table 1) and collect the eluate fractions. Continue the elution process, collecting fractions until the A&#8322;&#8328;&#8320; of the eluate drops below 0.01.</li>
		<li>Process Monitoring and Analysis: Collect samples from key purification stages&#8212;including the supernatant, precipitate, flow-through, wash fractions, and elution fractions. Analyze them by SDS-PAGE to assess purification efficiency (Supplementary Figure 1).</li>
		<li>Dialysis for Buffer Exchange: Transfer the pooled eluate into dialysis tubing and dialyze it against Dialysis Buffer (Supplementary Table 1) at 4 &#176;C with gentle stirring overnight.</li>
		<li>Ultrafiltration and concentration: Concentrate the dialyzed solution using an ultrafiltration device to a final MSP concentration of 2&#8211;5 mg/mL. The purified and concentrated MSP is now ready for use in membrane protein nanodisc assembly.</li>
	</ol>
	</li>
</ol>
3. Activation of Bio-Beads
<ol>
	<li>Weighing
	<ol>
		<li>Weigh approximately 250 mg of Bio-Beads into a 50 mL centrifuge tube using an analytical balance.</li>
	</ol>
	</li>
	<li>Methanol washes
	<ol>
		<li>Add 25&#8211;30 mL of methanol to the tube. Thoroughly rotate or vortex the mixture in the dark, then incubate for 20 min. Carefully discard the supernatant after incubation.</li>
		<li>Repeat this methanol washing step three times in total. 
		NOTE: Due to the volatility of methanol, this procedure must be performed in a fume hood with appropriate personal protective equipment. Collect used methanol and treat it as biological waste liquid.</li>
	</ol>
	</li>
	<li>Aqueous washes
	<ol>
		<li>Add 25&#8211;30 mL ddH2O to the beads. Shake or rotate the tube to mix and incubate for 20 min.</li>
		<li>Discard the supernatant. Repeat this aqueous washing step three times.</li>
	</ol>
	</li>
	<li>Equilibration with LSB
	<ol>
		<li>Add 25&#8211;30 mL of detergent-free LSB to the beads. Shake or rotate the tube on a rotating table to mix and incubate for 20 min.</li>
		<li>Discard the supernatant. Repeat this LSB equilibration step three times to fully condition the beads.</li>
	</ol>
	</li>
	<li>Aliquoting and storage
	<ol>
		<li>Divide the activated Bio-Beads equally into three 1.5 mL microcentrifuge tubes. Ensure the beads remain moistened with a small amount of LSB.</li>
		<li>Wrap the tubes in aluminum foil to protect from light and store them on ice for immediate use.</li>
	</ol>
	</li>
</ol>
4. Assembling the nanodisc
<ol>
	<li>Preparation of lipid and MSP
	<ol>
		<li>Lipid Stock Clarification: Retrieve one aliquot of the pre-prepared lipid stock and thaw it. Immediately centrifuge the tube at&#160;7,000 x g for 5 s at 4 &#176;C to collect the contents at the bottom.</li>
		<li>Ultrasonicate in a water bath and monitor the clarity and temperature every 5&#8211;10 min to prevent overheating. Perform sonication at 60 % amplitude. Ultrasonicate the lipid stock until the lipid stock becomes clear and transparent.</li>
		<li>MSP Thawing: Thaw 1 aliquot of the prepared MSP solution in an ice-water bath.</li>
	</ol>
	</li>
	<li>Prepare the buffer according to the stoichiometric ratio.
	<ol>
		<li>Quantify the Membrane Protein:&#160;Isolate the purified membrane protein (Task2), solubilized in 0.025% DDM/0.005% CHS, by size-exclusion chromatography (SEC). Inject a sample volume of 500 &#181;L at a flow rate of 0.5 mL/min onto a Superose 6 Increase column (Cytiva). Collect the peak eluting at 14.8 mL and concentrate it (Figure 1). Determine the protein concentration using a NanoDrop.</li>
		<li>Calculate the Protein Molar Amount:&#160;Calculate the molar amount (n) of the target membrane protein using the formula: n = C &#215; V, where&#160;C&#160;is the molar concentration and&#160;V&#160;is the volume.</li>
		<li>Determine the MSP and Lipid Volumes:&#160;Calculate the required volumes of MSP and lipid stock solutions based on a molar stoichiometric ratio of target protein : MSP : lipid = 1 : 5 : 250.</li>
		<li>Prepare the Master Mix:&#160;Based on the calculation results, combine the corresponding volumes of lipid and MSP to prepare a 300 &#181;L master mix. 
		NOTE: Ensure detergent is included in this mix to maintain a concentration consistent with that in the protein solution, thereby preventing premature aggregation.</li>
	</ol>
	</li>
	<li>Detergent removal and nanodisc assembly
	<ol>
		<li>Initial Incubation with Reconstitution Mix: Incubate the prepared mix buffer (Supplementary Table 1) on ice for 10 min. Then, slowly add 50 &#181;L of the mix to the concentrated protein solution, mixing gently by pipetting to ensure homogeneity.</li>
		<li>Equilibration of the Protein-Lipid Mixture: Place the combined mixture on ice for 1 h. Gently invert the tube every 15 min to mix thoroughly.</li>
		<li>First Incubation with Bio-Beads: Briefly centrifuge the mixture at&#160;7,000 x g for 5 s at 4 &#176;C to collect the protein solution at the bottom of the tube. Take one aliquot of the pre-activated Bio-Beads, aspirate the residual LSB, add the protein solution to the beads, and incubate with rotation in the dark for 1 h to prevent light-induced degradation of the target protein.</li>
		<li>Second Incubation with Bio-Beads: Centrifuge the mixture at&#160;7,000 x g for 5 s at 4 &#176;C and carefully transfer the supernatant to a second aliquot of Bio-Beads (from which the LSB has also been aspirated) and mix well by gentle inversion in the dark for 4&#8211;8 h or overnight.</li>
		<li>Final Incubation with Bio-Beads: Retrieve the protein solution after overnight incubation, centrifuge it briefly, and transfer the supernatant to the third and final portion of pre-activated Bio-Beads. Incubate with rotation at 4 &#176;C in the dark for 1 h.</li>
		<li>Following a brief centrifugation, transfer the supernatant to a clean microcentrifuge tube and centrifuge at 17,000 x g at 4 &#176;C for 10 min to remove any aggregates. 
		NOTE: Dispose of spent Bio-Beads as biological waste.</li>
		<li>Purification of Nanodiscs: Purify the nanodisc-Task2 complexes by size-exclusion chromatography (SEC): Inject a sample volume of 500 &#181;L at a flow rate of 0.5 mL/min onto a Superose 6 Increase column and collect the peak eluting at 15 mL (Figure 2).</li>
		<li>Concentration of the Final Product: Pool the peak fractions from the SEC elution and concentrate them to a final nanodisc-Task2 concentration of 1.2&#8211;1.5 mg/mL, suitable for cryo-EM sample preparation.</li>
	</ol>
	</li>
</ol>
5. Verify that the nanodisc assembly is successful
<ol>
	<li>SDS-PAGE Analysis: Aliquot samples from the SEC-purified fractions.
	<ol>
		<li>Analyze the samples using 12% SDS-PAGE to confirm the presence of key components (e.g., MSP and target membrane protein), which indicates successful nanodisc assembly (Figure 3).</li>
	</ol>
	</li>
	<li>Cryo-EM Sample Preparation and Analysis.
	<ol>
		<li>Concentrate the successfully assembled sample to&#160;a final concentration of 1.2&#8211;1.5 mg/mL for cryo-EM sample preparation.</li>
		<li>Begin by centrifuging the sample at 17,000 x&#160;g and 4&#160;&#176;C for 10&#160;min. Deposit a 3&#160;&#181;L droplet onto freshly glow-discharged Quantifoil R1.2/1.3 gold grids (300 mesh, holey carbon).</li>
		<li>Incubate at 4&#160;&#176;C in 100% humidity for 5&#160;s. Blot the grid for 4&#160;s in a Vitrobot Mark IV and plunge-freeze in liquid ethane maintained at liquid nitrogen temperature.</li>
		<li>Collect super-resolution movie stacks using EPU software on a Titan Krios G4 cryo-EM operating at 300 kV and equipped with a Falcon G4i direct electron detector.</li>
		<li>Set the exposure time to 2.78 s, corresponding to a pixel size of 0.73 &#197; (165k &#215;) and a total accumulated dose of approximately 50 e&#8315;/&#197;2 per EER movie stack.</li>
	</ol>
	</li>
</ol>

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<li>Tao, X., Zhao, C., MacKinnon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Membrane+protein+isolation+and+structure+determination+in+cell-derived+membrane+vesicles%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.S.A%22%5BJournal%5D)">Membrane protein isolation and structure determination in cell-derived membrane vesicles.</a> Proc. Natl. Acad. Sci. U.S.A. 120 (18), e2302325120(2023).</li>
<li>Zhang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((One-step+construction+of+circularized+nanodiscs+using+SpyCatcher-SpyTag%5BTitle%5D)+AND+%22Nat.+Commun%22%5BJournal%5D)">One-step construction of circularized nanodiscs using SpyCatcher-SpyTag.</a> Nat. Commun. 12 (1), 5451(2021).</li>
<li>Ren, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((DeFrND%3A+detergent-free+reconstitution+into+native+nanodiscs+with+designer+membrane+scaffold+peptides%5BTitle%5D)+AND+%22Nat.+Commun%22%5BJournal%5D)">DeFrND: detergent-free reconstitution into native nanodiscs with designer membrane scaffold peptides.</a> Nat. Commun. 16 (1), 7973(2025).</li>
</ol>

The authors have no conflicts of interest to disclose.

The method is presented for reconstituting detergent-solubilized membrane proteins into nanodiscs via detergent removal, thereby providing a more native lipid bilayer environment. The success of this assembly process hinges on several key factors. Foremost among these is the precise stoichiometric ratio among the membrane protein, MSP, and lipids. Improper stoichiometric ratios can lead to protein aggregation or unsuccessful assembly. For example, the first attempt to assemble nanodiscs using a 1:2:50 ratio resulted in t...

Membrane proteins play a crucial role in cellular signal transduction, metabolite transport, maintaining cell structural integrity, and immune responses. Dysregulation of membrane proteins is implicated in various diseases, making them prominent targets for drug development1. The advent of cryo-electron microscopy (cryo-EM) has enabled structural studies of large and dynamic membrane proteins2. A main challenge in obtaining high-resolution structures lies in extracting these proteins from their native lipid bilayers while preserving their native conformations and biochemical functions. Moreover, the highly dynamic conformation of membrane proteins and the complexity of antibody screening pose additional obstacles to targeted drug discovery3,4. Traditional methods for membrane protein extraction often employ detergents to mimic the properties of the hydrophobic lipid bilayer. However, this approach often disrupts membrane protein stability, leading to aggregation or partial unfolding5. Additionally, detergents may remove essential lipid molecules that are critical for stabilizing the protein conformation, potentially altering protein conformation or even impairing function, thereby limiting their application in antibody screening and other downstream applications6.
Nanodisc technology was first introduced by Sligar and colleagues7. This self-assembling structure is typically formed by apolipoprotein A1 (ApoA1) derivatives, namely MSPs, together with synthetic or natural lipids, providing a stable environment for membrane proteins7,8,9. The MSP forms a disc-shaped structure by surrounding the hydrophobic edges of the lipid bilayer. This structure has a hydrophobic surface that interacts with the acyl lipid tails, stabilizing the membrane protein, and a hydrophilic surface facing outward, allowing the membrane protein to remain soluble in aqueous solutions10. Nanodiscs serve as a versatile platform for membrane protein studies. By preserving purified membrane proteins in a near-native lipid environment, they overcome the limitations of traditional detergent-based methods and maintain the proteins&#8217; conformation and biological function in vitro. Consequently, this method has become a powerful tool for structural studies of membrane proteins. Compared with traditional detergent environments, the lipid bilayer in nanodiscs provides structural support for membrane proteins, stabilizing their native conformation&#8212;a feature crucial for proteins prone to inactivation, such as G protein-coupled receptors and ion channels11. Additionally, the highly uniform nature of nanodisc particles after assembly offers stronger support for obtaining high-quality cryo-EM data and reliable surface plasmon resonance/bio-layer interferometry kinetic data12.
Over the past decade, nanodisc technology has become a powerful tool for studying membrane proteins in natural lipid environments. For instance, Vilela and colleagues13 have established a systematic framework for nanodisc assembly by optimizing the reconstitution conditions and verifying the quality of the final components. Specifically, they combined size exclusion chromatography (SEC) with quantitative indicators (including assembly efficiency and peak symmetry) to objectively assess the homogeneity of the samples13. Currently, nanodiscs have become a versatile platform. By using single-particle cryo-EM to detect the assembled nanodisc-reconstituted proteins, high-resolution structures of membrane proteins can be obtained, and their applicability has been firmly established. The double-ring density feature of the MSP band is the decisive structural evidence for successful binding10,14,15. Furthermore, nanodiscs serve as a valuable tool for both the functional analysis of membrane proteins in lipid bilayer environments and their application in solution nuclear magnetic resonance studies16,17.
This protocol describes a method for assembling the human Task2 membrane protein into nanodiscs. Human Task2 is a pH-gated member of the two-pore domain K+ (K2P) channel family18,19. Like other K2P channels, Task2 is a domain-swapped homodimer, with each protomer chain containing four transmembrane-spanning helices (TM1&#8211;TM4), two reentrant pore helices (PH1 and PH2), two selectivity filters (SF1 and SF2), and two extracellular cap-forming helices (CH1 and CH2). The target protein was a truncated form of Task2 lacking the C-terminal 166 amino acids&#8212;a region predicted to be largely unstructured&#8212;which was found to improve protein expression and biochemical stability. The molecular weight of recombined Task2 is approximately 72 kDa with a GFP tag. It had been solubilized and purified using a detergent beforehand. It was combined with lipids and MSP at a precisely calculated stoichiometric ratio. Following detergent removal using Bio-Beads, the sample was further purified by size-exclusion chromatography (SEC) to obtain a preparation suitable for cryo-EM. Two-dimensional (2D) class averages were then generated from single-particle cryo-EM data to validate sample integrity.

<table><tbody><tr><td>Bio-beads</td><td>Bio-rad</td><td>1523920</td><td></td></tr><tr><td>Cholesteryl hemisuccinate</td><td>Anatrace</td><td>CH210</td><td></td></tr><tr><td>Chloroform</td><td>Sinoreagent</td><td></td><td>Use in a fume hood</td></tr><tr><td>DOPE</td><td>Avanti</td><td>850725P</td><td></td></tr><tr><td>Methanol</td><td>Sinoreagent</td><td></td><td></td></tr><tr><td>n-Dodecyl-beta-D-maltoside</td><td>Anatrace</td><td>D310</td><td></td></tr><tr><td>n-pentane</td><td>Adamas</td><td></td><td>Use in a fume hood</td></tr><tr><td>POPC</td><td>Avanti</td><td>850457P</td><td></td></tr><tr><td>POPS</td><td>Avanti</td><td>840034P</td><td></td></tr><tr><td>SEC</td><td>Bio-rad</td><td></td><td>Follow manufacturer&#039;s protocol for proper use</td></tr><tr><td>SnakeSkin Dialysis Tubing</td><td>Thermo</td><td>68011</td><td></td></tr><tr><td>Superose&amp;nbsp;6 Increase10/300 GL</td><td>Cytiva</td><td>29091596</td><td></td></tr><tr><td>Ultrafiltration Spin Columns Amicon Ultra-15</td><td>Millipore</td><td>UFC905008</td><td></td></tr></tbody></table>

a technique for stabilizing membrane proteins in nanodiscs

A Technique for Stabilizing Membrane Proteins in Nanodiscs

Many membrane proteins exhibit poor stability, loss of activity, and sample heterogeneity when extracted in detergent-based environments, posing substantial challenges for high-resolution cryo-electron microscopy analysis. The nanodisc technology addresses these limitations by employing membrane scaffold proteins (MSPs) and phospholipids to reconstitute target proteins into a native-like lipid bilayer. In this study, the human potassium channel Task2 was expressed, solubilized, and purified in the presence of appropriate detergents prior to nanodisc assembly. The resulting protocol establishes a reproducible workflow&#160;for Task2 nanodisc reconstitution, as demonstrated by key validation steps: a clear shift in size exclusion chromatography (SEC) profiles confirming particle enlargement, co-migration of Task2 and MSP on SDS-PAGE, and well-defined 2D class averages with visible secondary structure features. An initial 3D reconstruction further confirms the integrity of the nanodisc-embedded protein. By improving sample purity, homogeneity, and particle quality, this approach provides a robust platform for high-resolution structural studies of Task2 and offers a methodological framework applicable to other membrane proteins.

Successful preparation of the lipid stock was indicated by the formation of a clear, transparent solution after sonication. For the MSP, nickel-affinity chromatography yielded a high-purity product at approximately 3 mg/mL. The assembly of nanodiscs was confirmed by SEC, which showed a characteristic elution profile (Figure 2). Compared with the Task2 SEC profile obtained prior to nanodisc assembly (Figure 1), the protein peak shifts to a higher elution volume (later elution) after assembly....

This study outlines the procedure for reconstituting the membrane protein TWIK-related acid-sensitive K+ channel 2 (Task2) into nanodiscs. The successful assembly was confirmed by single-particle cryo-electron microscopy, which yielded well-defined two-dimensional class averages.

Many membrane proteins exhibit poor stability, loss of activity, and sample heterogeneity when extracted in detergent-based environments, posing ...

a-technique-for-stabilizing-membrane-proteins-in-nanodiscs

Research

JoVE Journal

Biology

Nanodiscs are small discoid particles that incorporate membrane proteins into a small patch of phospholipid bilayer.  We provide a visual protocol that shows the step-by-step incorporation of the MalFGK2 transporter into a disc.

A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles

β-barrel outer membrane proteins (OMPs) serve many functions within the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts. Here, we hope to alleviate a known bottleneck in structural studies by presenting protocols for the production of β-barrel OMPs in sufficient quantities for structure determination by X-ray crystallography or NMR spectroscopy.

From Constructs to Crystals &#8211; Towards Structure Determination of &#946;-barrel Outer Membrane Proteins

We present a protocol to functionalize glass with nanometric protein patches surrounded by a fluid lipid bilayer. These substrates are compatible with advanced optical microscopy and are expected to serve as platform for cell adhesion and migration studies.

Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

Many proteins perform their function when attached to membrane surfaces. The binding of extrinsic proteins on nanodisc membranes can be indirectly imaged by transmission electron microscopy. We show that the characteristic stacking (rouleau) of nanodiscs induced by the negative stain sodium phosphotungstate is prevented by the binding of extrinsic protein.

Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy

This protocol demonstrates how to obtain a low-resolution ab initio model and structural details of a detergent-solubilized membrane protein in solution using small-angle neutron scattering with contrast-matching of the detergent.

Contrast-Matching Detergent in Small-Angle Neutron Scattering Experiments for Membrane Protein Structural Analysis and Ab Initio Modeling

We present a method that combines membrane protein purification and reconstitution into peptidiscs in a single chromatographic step. Biotinylated scaffolds are used for direct surface attachment and measurement of protein-ligand interactions via biolayer interferometry.

PeptiQuick, a One-Step Incorporation of Membrane Proteins into Biotinylated Peptidiscs for Streamlined Protein Binding Assays

Presented here is a protocol for the determination of oligomeric state of membrane proteins that utilizes a native cell membrane nanoparticle system in conjunction with electron microscopy.

Native Cell Membrane Nanoparticles System for Membrane Protein-Protein Interaction Analysis

Co-translational insertion into pre-formed nanodiscs makes it possible to study cell-free synthesized membrane proteins in defined lipid environments without contact with detergents. This protocol describes the preparation of essential system components and the critical parameters for improving expression efficiency and sample quality.

Co-Translational Insertion of Membrane Proteins into Preformed Nanodiscs

Here, a protocol is described for generating light-regulated and reversible protein patterns with high spatiotemporal precision at artificial lipid membranes. The method consists of the localized photoactivation of the protein iLID (improved light-inducible dimer) immobilized on model membranes that, under blue light, binds to its partner protein Nano (wild-type SspB).

Dynamic Light-Induced Protein Patterns at Model Membranes

We present a protocol for reconstituting membrane proteins and encapsulating enzymes and other water-soluble components in lipid vesicles of sub-micrometer and micrometer size.

Construction of Out&#45;of&#45;Equilibrium Metabolic Networks in Nano&#45; and Micrometer&#45;Sized Vesicles

Learn a robust protocol for reconstituting the human potassium channel Task2 into nanodiscs using membrane scaffold proteins and phospholipids. This method enables high-resolution cryo-EM structural analysis.

A Technique for Stabilizing Membrane Proteins in Nanodiscs

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