Assembled Nanodiscs


Nanodisc

Assembled nanodiscs

Assembled nanodiscs are available as part of a collaboration with the Institute of Biophysics at the University of Frankfurt. They are conveniently aliquoted in 50 µL solutions of 0.5 mM concentration. End concentrations in the cell-free reaction range from 20-80 µM. Bacterial and eucaryotic membrane proteins can be expressed (Fig 1 and 2), activity may depend on the lipid composition of the assembled nanodisc. Alternatively, nanodiscs can be assembled from the pre-aliquoted components provided in our Nanodisc Assembly kits.
Please note that assembled nanodiscs are intended for use in cell-free expression reactions only. They are not recommended for the reconstitution of membrane proteins already solubilized in detergent, or for the solubilization of proteins from membranes.

Available assembled nanodisc variations offer:

Nanodisc MSP1D1-His_DMPC was successfully used in the following publication:
Guo, S.. et al. Expressing Biologically Active Membrane Proteins in a Cell-Free Transcription-Translation Platform.
bioRxiv preprint Jan 30, 2017; doi: http://dx.doi.org/10.1101/104455

Assembles nanodiscs in detail

MraY_in_nanodiscs
Fig. 1: Stability and specific activity of E. coli MraY translocase in different lipid environments. The protein was cell-free synthesized in E. coli lysates in presence of pre-assembled MSP1E3D1 nanodiscs containing the indicated lipids. The topology of the membrane integrated MraY enzyme is indicated in the inset. MraY catalyses the lipid-I precursor formation of bacterial cell wall biosynthesis. The figure shows the lipid-I formation in nmol per µg catalyzed by the corresponding MraY/nanodisc samples (Roos et al., (2012) BBA 1818, 3098-3106). The activity of the enzyme strongly depends on anionic phosphatidylglycerol type lipids and was best with the shorter myristoyl fatty acid chains (DMPG). Data kindly provided by Frank Bernhard, University Frankfurt.
GPCR_in_nanocisc;g--protein-coupled_receptors_nanodisc
Fig. 2. Differential ligand binding activity of the human endothelin A (ETA) and endothelin B (ETB) receptors in MSP1E3D1 (DMPC) nanodiscs. The G protein-coupled receptors (GPCRs) ETA and ETB were cell-free synthesized in E. coli lysates in presence of pre-assembled MSP1E3D1 nanodiscs containing the lipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). The resulting receptor/nanodisc samples were analyzed for their characteristic binding affinities to four different peptide ligands by SPR (Proverbio et al., (2013) BBA 1828, 2182-2192). Both GPCRs have high affinity to the natural ligand ET-1 while the affinities to the other three ligands is receptor specific. The characteristic differential ligand binding profile of the cell-free synthesized GPCR/nanodisc samples was obtained and in good agreement with data from the literature. Data kindly provided by Frank Bernhard, University Frankfurt.

Nanodisc MSP proteins from Cube Biotech were successfully used in the following publications:

 ProteinYearAuthor
β2AR/β1AR 2017 Guo S, Vaish A., Chen Q., Murray R1.
Tse6 2019 Quentin D., Ahmad S., Shanthamoorthy P., Mougous J., Whitney J., Raunser S.2
K+ channel proteins 2018  Khoury M., Winterstein T., Weber W., Stein V., Schlaak H., Thiel G.3
KcvNTS 2018 Winterstein L.-M., Kukovetz K., Rauh O., Turman D., Braun C., Moroni A., Schroeder I., Thiel G.4

 

Features

Purpose Forms nanodisc structures in combination with phospholipids
Specifity Stabilization of membrane proteins
Characteristics MSP1D1, MSP1E3D1 or MSP2N2 wild-type protein with DMPC, POPC or DMPG
Resulting nanodisc diameter: ca. 7-17 nm
Purity > 90 %
Component Assembled nanodisc with DMPC, POPC or DMPG
Required equipment
 
  • Micropipettor and Micropipetting tips
  • Gel filtration column
  • FPLC instrument with integrated UV detector and fraction collector
  • Magnetic stirrer
  • Centrifuge for 2 mL microtubes
  • 2 mL microtubes
  • Palmitoyl-oleoyl-phosphatidylcholine (POPC)
  • Dimyristoyl-glycero-phosphocholine (DMPC) or other suitable phospholipid or phospholipid mixture
  • Sodium cholate
  • Biobeads SM-2
  • Single use syringe
  • End-over-end shaker
  • Single use needle
  • SDS PAGE reagents and equipment
  • Protein concentrator
  • 0.45 micron filter
  • Optional: Western Blot reagents and equipment and PentaHis Antibody

Applications

All protocols are also avaible as PDF-Files on the Protocols & Datasheets page.
   
A.DMPC: Assembly of nanodiscs for use in cell-free expression using MSP1D1-His protein and DMPC phospholipids
 
 
  1. Prepare a 100 mM sodium cholate solution by adding 464 µl of ND Buffer B to the 20 mg aliquot (clear capped plastic vial).
  2. Resuspend the contents of the blue-marked brown glass vial containing 4.65 mg DMPC phospholipid with 137 µl of the 100 mM sodium cholate/ND Buffer B solution prepared in step 1. Note: Open the glass vial carefully as it contains a microglass tube held in position by a spring.
  3. Incubate the solution obtained in step 2 for 30 min at 37°C on a thermoshaker, or alternatively mix by flicking/inverting the tube every 10 min.
  4. Resuspend contents of the blue-capped plastic vial containing the lyophilized MSP1D1-His protein in 500 µl double distilled water.
  5. Add 250 µl Buffer A and 250 µl of the 100 mM sodium cholate /ND Buffer B solution to the resuspended protein solution. Briefly spin the solution down. Keep on ice.
  6. Add the entire volume of the solution obtained in step 3 containing DMPC and sodium cholate/ND Buffer B to the resuspended protein solution obtained in step 5.
  7. Incubate the mix obtained in step 6 for 20 min at 4°C, then incubate for 20 min at 37°C.
  8. Repeat step 7 twice, for a total incubation time of 2 h.
  9. . Fill the nanodisc mix into a dialysis tube of 3-8 kDa cutoff pore size and dialyse for two days at 4°C against ND Buffer A. Exchange the buffer about 2-4 times during this period.
  10. . Apply the nanodisc mix on a gel filtration column. Monitor absorbance at 280 nm.
  11. Collect fractions of ca. 500 µL size, take an aliquot of 20 µL, add 5 µL of 5xSDS-PAGE buffer and analyze the samples by SDS PAGE. MSP proteins have an apparent molecular mass of around 20 kDa.
  12. Concentrate the elution fractions which contain the nanodiscs using protein concentrators to 50 µl and store them at -20°C.

 
   
B.Assembly of nanodiscs for use in cell-free expression using MSP1D1-His protein and POPC phospholipids
 
 
  1. Using one of the 20 mg sodium cholate aliquots (clear capped plastic vial), prepare a 200 mM sodium cholate solution by adding 232 µl of ND Buffer B.
  2. Using the other 20 mg sodium cholate aliquot, prepare a 100 mM sodium cholate solution by adding 464 µl of ND Buffer B.
  3. Resuspend the contents of the blue-marked brown glass vial containing 3.58 mg POPC phospholipid with 94 µl of the 200 mM sodium cholate/ND Buffer B solution prepared in step 1. Note: Open the glass vial carefully as it contains a microglass tube held in position by a spring.
  4. Incubate the solution obtained in step 3 for 30 min on ice. Mix by flicking or inverting the tube every 10 min.
  5. Resuspend contents of the blue-capped plastic vial containing the lyophilized MSP1D1-His protein in 500 µl double distilled water
  6. Add 250 µl Buffer A and 250 µl of the 100 mM sodium cholate /ND Buffer B solution to the resuspended protein solution. Briefly spin the solution down. Keep on ice.
  7. Add the entire volume of the solution obtained in step 4 containing POPC and sodium cholate/ND Buffer B to the resuspended protein solution obtained in step 6.
  8. Incubate the mix obtained in step 6 for 1 h on ice.
  9. Fill the nanodisc mix into a dialysis tube of 3-8 kDa cutoff pore size and dialyse for two days at 4°C against ND Buffer A. Exchange the buffer about 2-4 times during this period.
  10. Apply the nanodisc mix on a gel filtration column. Monitor absorbance at 280 nm.
  11. Collect fractions of ca. 500 µL size, take an aliquot of 20 µL, add 5 µL of 5xSDS-PAGE buffer and analyze the samples by SDS PAGE. MSP proteins have an apparent molecular mass of around 20 kDa.
  12. Concentrate the elution fractions which contain the nanodiscs using protein concentrators to 50 µl and store them at -20°C.

References

  1. Guo, Shaobin, et al. "Expressing Biologically Active Membrane Proteins in a Cell-Free Transcription-Translation Platform." (2017).
  2. Quentin, Dennis, et al. "Mechanism of loading and translocation of type VI secretion system effector Tse6." Nature microbiology 3.10 (2018): 1142.
  3. El Khoury, Mario, et al. "Photolithographic Fabrication of Micro Apertures in Dry Film Polymer Sheets for Channel Recordings in Planar Lipid Bilayers." The Journal of membrane biology 252.2-3 (2019): 173-182.
  4. Winterstein, Laura-Marie, et al. "Reconstitution and functional characterization of ion channels from nanodiscs in lipid bilayers." The Journal of general physiology 150.4 (2018): 637-646.
 
Nanodisc

Assembled nanodiscs

Assembled nanodiscs are available as part of a collaboration with the Institute of Biophysics at the University of Frankfurt. They are conveniently aliquoted in 50 µL solutions of 0.5 mM concentration. End concentrations in the cell-free reaction range from 20-80 µM. Bacterial and eucaryotic membrane proteins can be expressed (Fig 1 and 2), activity may depend on the lipid composition of the assembled nanodisc. Alternatively, nanodiscs can be assembled from the pre-aliquoted components provided in our Nanodisc Assembly kits.
Please note that assembled nanodiscs are intended for use in cell-free expression reactions only. They are not recommended for the reconstitution of membrane proteins already solubilized in detergent, or for the solubilization of proteins from membranes.

Available assembled nanodisc variations offer:

Nanodisc MSP1D1-His_DMPC was successfully used in the following publication:
Guo, S.. et al. Expressing Biologically Active Membrane Proteins in a Cell-Free Transcription-Translation Platform.
bioRxiv preprint Jan 30, 2017; doi: http://dx.doi.org/10.1101/104455

Assembles nanodiscs in detail

MraY_in_nanodiscs
Fig. 1: Stability and specific activity of E. coli MraY translocase in different lipid environments. The protein was cell-free synthesized in E. coli lysates in presence of pre-assembled MSP1E3D1 nanodiscs containing the indicated lipids. The topology of the membrane integrated MraY enzyme is indicated in the inset. MraY catalyses the lipid-I precursor formation of bacterial cell wall biosynthesis. The figure shows the lipid-I formation in nmol per µg catalyzed by the corresponding MraY/nanodisc samples (Roos et al., (2012) BBA 1818, 3098-3106). The activity of the enzyme strongly depends on anionic phosphatidylglycerol type lipids and was best with the shorter myristoyl fatty acid chains (DMPG). Data kindly provided by Frank Bernhard, University Frankfurt.
GPCR_in_nanocisc;g--protein-coupled_receptors_nanodisc
Fig. 2. Differential ligand binding activity of the human endothelin A (ETA) and endothelin B (ETB) receptors in MSP1E3D1 (DMPC) nanodiscs. The G protein-coupled receptors (GPCRs) ETA and ETB were cell-free synthesized in E. coli lysates in presence of pre-assembled MSP1E3D1 nanodiscs containing the lipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). The resulting receptor/nanodisc samples were analyzed for their characteristic binding affinities to four different peptide ligands by SPR (Proverbio et al., (2013) BBA 1828, 2182-2192). Both GPCRs have high affinity to the natural ligand ET-1 while the affinities to the other three ligands is receptor specific. The characteristic differential ligand binding profile of the cell-free synthesized GPCR/nanodisc samples was obtained and in good agreement with data from the literature. Data kindly provided by Frank Bernhard, University Frankfurt.

Nanodisc MSP proteins from Cube Biotech were successfully used in the following publications:

 ProteinYearAuthor
β2AR/β1AR 2017 Guo S, Vaish A., Chen Q., Murray R1.
Tse6 2019 Quentin D., Ahmad S., Shanthamoorthy P., Mougous J., Whitney J., Raunser S.2
K+ channel proteins 2018  Khoury M., Winterstein T., Weber W., Stein V., Schlaak H., Thiel G.3
KcvNTS 2018 Winterstein L.-M., Kukovetz K., Rauh O., Turman D., Braun C., Moroni A., Schroeder I., Thiel G.4

 

Features

Purpose Forms nanodisc structures in combination with phospholipids
Specifity Stabilization of membrane proteins
Characteristics MSP1D1, MSP1E3D1 or MSP2N2 wild-type protein with DMPC, POPC or DMPG
Resulting nanodisc diameter: ca. 7-17 nm
Purity > 90 %
Component Assembled nanodisc with DMPC, POPC or DMPG
Required equipment
 
  • Micropipettor and Micropipetting tips
  • Gel filtration column
  • FPLC instrument with integrated UV detector and fraction collector
  • Magnetic stirrer
  • Centrifuge for 2 mL microtubes
  • 2 mL microtubes
  • Palmitoyl-oleoyl-phosphatidylcholine (POPC)
  • Dimyristoyl-glycero-phosphocholine (DMPC) or other suitable phospholipid or phospholipid mixture
  • Sodium cholate
  • Biobeads SM-2
  • Single use syringe
  • End-over-end shaker
  • Single use needle
  • SDS PAGE reagents and equipment
  • Protein concentrator
  • 0.45 micron filter
  • Optional: Western Blot reagents and equipment and PentaHis Antibody

Applications

All protocols are also avaible as PDF-Files on the Protocols & Datasheets page.
   
A.DMPC: Assembly of nanodiscs for use in cell-free expression using MSP1D1-His protein and DMPC phospholipids
 
 
  1. Prepare a 100 mM sodium cholate solution by adding 464 µl of ND Buffer B to the 20 mg aliquot (clear capped plastic vial).
  2. Resuspend the contents of the blue-marked brown glass vial containing 4.65 mg DMPC phospholipid with 137 µl of the 100 mM sodium cholate/ND Buffer B solution prepared in step 1. Note: Open the glass vial carefully as it contains a microglass tube held in position by a spring.
  3. Incubate the solution obtained in step 2 for 30 min at 37°C on a thermoshaker, or alternatively mix by flicking/inverting the tube every 10 min.
  4. Resuspend contents of the blue-capped plastic vial containing the lyophilized MSP1D1-His protein in 500 µl double distilled water.
  5. Add 250 µl Buffer A and 250 µl of the 100 mM sodium cholate /ND Buffer B solution to the resuspended protein solution. Briefly spin the solution down. Keep on ice.
  6. Add the entire volume of the solution obtained in step 3 containing DMPC and sodium cholate/ND Buffer B to the resuspended protein solution obtained in step 5.
  7. Incubate the mix obtained in step 6 for 20 min at 4°C, then incubate for 20 min at 37°C.
  8. Repeat step 7 twice, for a total incubation time of 2 h.
  9. . Fill the nanodisc mix into a dialysis tube of 3-8 kDa cutoff pore size and dialyse for two days at 4°C against ND Buffer A. Exchange the buffer about 2-4 times during this period.
  10. . Apply the nanodisc mix on a gel filtration column. Monitor absorbance at 280 nm.
  11. Collect fractions of ca. 500 µL size, take an aliquot of 20 µL, add 5 µL of 5xSDS-PAGE buffer and analyze the samples by SDS PAGE. MSP proteins have an apparent molecular mass of around 20 kDa.
  12. Concentrate the elution fractions which contain the nanodiscs using protein concentrators to 50 µl and store them at -20°C.

 
   
B.Assembly of nanodiscs for use in cell-free expression using MSP1D1-His protein and POPC phospholipids
 
 
  1. Using one of the 20 mg sodium cholate aliquots (clear capped plastic vial), prepare a 200 mM sodium cholate solution by adding 232 µl of ND Buffer B.
  2. Using the other 20 mg sodium cholate aliquot, prepare a 100 mM sodium cholate solution by adding 464 µl of ND Buffer B.
  3. Resuspend the contents of the blue-marked brown glass vial containing 3.58 mg POPC phospholipid with 94 µl of the 200 mM sodium cholate/ND Buffer B solution prepared in step 1. Note: Open the glass vial carefully as it contains a microglass tube held in position by a spring.
  4. Incubate the solution obtained in step 3 for 30 min on ice. Mix by flicking or inverting the tube every 10 min.
  5. Resuspend contents of the blue-capped plastic vial containing the lyophilized MSP1D1-His protein in 500 µl double distilled water
  6. Add 250 µl Buffer A and 250 µl of the 100 mM sodium cholate /ND Buffer B solution to the resuspended protein solution. Briefly spin the solution down. Keep on ice.
  7. Add the entire volume of the solution obtained in step 4 containing POPC and sodium cholate/ND Buffer B to the resuspended protein solution obtained in step 6.
  8. Incubate the mix obtained in step 6 for 1 h on ice.
  9. Fill the nanodisc mix into a dialysis tube of 3-8 kDa cutoff pore size and dialyse for two days at 4°C against ND Buffer A. Exchange the buffer about 2-4 times during this period.
  10. Apply the nanodisc mix on a gel filtration column. Monitor absorbance at 280 nm.
  11. Collect fractions of ca. 500 µL size, take an aliquot of 20 µL, add 5 µL of 5xSDS-PAGE buffer and analyze the samples by SDS PAGE. MSP proteins have an apparent molecular mass of around 20 kDa.
  12. Concentrate the elution fractions which contain the nanodiscs using protein concentrators to 50 µl and store them at -20°C.

References

  1. Guo, Shaobin, et al. "Expressing Biologically Active Membrane Proteins in a Cell-Free Transcription-Translation Platform." (2017).
  2. Quentin, Dennis, et al. "Mechanism of loading and translocation of type VI secretion system effector Tse6." Nature microbiology 3.10 (2018): 1142.
  3. El Khoury, Mario, et al. "Photolithographic Fabrication of Micro Apertures in Dry Film Polymer Sheets for Channel Recordings in Planar Lipid Bilayers." The Journal of membrane biology 252.2-3 (2019): 173-182.
  4. Winterstein, Laura-Marie, et al. "Reconstitution and functional characterization of ion channels from nanodiscs in lipid bilayers." The Journal of general physiology 150.4 (2018): 637-646.
 
Assembled Nanodiscs
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Nanodisc MSP1D1-His_DMPC (50 µL) Nanodisc MSP1D1-His_DMPC (50 µL)
50 microliter pre-assembled nanodisc, 0.5 mM concentrated, containing MSP1D1-His protein and DMPC. For use in cell-free reactions only. Product is shipped on dry ice - additional freight charges apply.  
Article number: 26311
Sales price: €361.80 * €402.00 *
Nanodisc MSP2N2-His_DMPC (50 µL) Nanodisc MSP2N2-His_DMPC (50 µL)
50 microliter pre-assembled nanodisc, 0.5 mM concentrated, containing MSP2N2-His protein and DMPC. For use in cell-free reactions only. Product is shipped on dry ice - additional freight charges apply.
Article number: 26371
Sales price: €443.70 * €493.00 *
Nanodisc MSP1D1-His_POPC (50 µL) Nanodisc MSP1D1-His_POPC (50 µL)
50 microliter pre-assembled nanodisc, 0.5 mM concentrated, containing MSP1D1-His protein and POPC. For use in cell-free reactions only. Product is shipped on dry ice - additional freight charges apply.    
Article number: 26313
Sales price: €361.80 * €402.00 *
Nanodisc MSP1D1-His_DMPG (50 µL) Nanodisc MSP1D1-His_DMPG (50 µL)
50 microliter pre-assembled nanodisc, 0.5 mM concentrated, containing MSP1D1-His protein and DMPG. For use in cell-free reactions only. Product is shipped on dry ice - additional freight charges apply.
Article number: 26315
Sales price: €361.80 * €402.00 *
Nanodisc MSP1D1 dH5-His_DMPC (50 µL) Nanodisc MSP1D1 dH5-His_DMPC (50 µL)
50 microliter pre-assembled nanodisc, 0.5 mM concentrated, containing MSP1D1 deltaH5-His protein and DMPC. For use in cell-free reactions only. Product is shipped on dry ice - additional freight charges apply.      
Article number: 26321
Sales price: €361.80 * €402.00 *
Nanodisc MSP1D1 dH5-His_POPC (50 µL) Nanodisc MSP1D1 dH5-His_POPC (50 µL)
50 microliter pre-assembled nanodisc, 0.5 mM concentrated, containing MSP1D1 deltaH5-His protein and POPC. For use in cell-free reactions only. Product is shipped on dry ice - additional freight charges apply.    
Article number: 26323
Sales price: €361.80 * €402.00 *
Nanodisc MSP1D1 dH5-His_DMPG (50 µL) Nanodisc MSP1D1 dH5-His_DMPG (50 µL)
50 microliter pre-assembled nanodisc, 0.5 mM concentrated, containing MSP1D1 deltaH5-His protein and POPC. For use in cell-free reactions only. Shipped on dry ice - additional freight cost apply.
Article number: 26325
Sales price: €361.80 * €402.00 *
Nanodisc MSP1D1_DMPC (50 µL) Nanodisc MSP1D1_DMPC (50 µL)
50 microliter pre-assembled nanodisc, 0.5 mM concentrated, containing MSP1D1 protein and DMPC. For use in cell-free reactions only. Shipped on dry ice - additional freight cost apply.
Article number: 26331
Sales price: €526.00 *
Nanodisc MSP1D1_POPC (50 µL) Nanodisc MSP1D1_POPC (50 µL)
50 microliter pre-assembled nanodisc, 0.5 mM concentrated, containing MSP1D1 protein and POPC. For use in cell-free reactions only. Shipped on dry ice - additional freight cost apply.
Article number: 26333
Sales price: €526.00 *
Nanodisc MSP1D1_DMPG (50 µL) Nanodisc MSP1D1_DMPG (50 µL)
50 microliter pre-assembled nanodisc, 0.5 mM concentrated, containing MSP1D1 protein and DMPG. FFor use in cell-free reactions only. Shipped on dry ice - additional freight cost apply.
Article number: 26335
Sales price: €526.00 *
Nanodisc MSP1D1 dH5_DMPC (50 µL) Nanodisc MSP1D1 dH5_DMPC (50 µL)
50 microliter pre-assembled nanodisc, 0.5 mM concentrated, containing MSP1D1 deltaH5 protein and DMPC. For use in cell-free reactions only. Shipped on dry ice - additional freight cost apply.
Article number: 26341
Sales price: €526.00 *
Nanodisc MSP1D1 dH5_POPC (50 µL) Nanodisc MSP1D1 dH5_POPC (50 µL)
50 microliter pre-assembled nanodisc, 0.5 mM concentrated, containing MSP1D1 deltaH5 protein and POPC. For use in cell-free reactions only. Shipped on dry ice - additional freight cost apply.
Article number: 26343
Sales price: €526.00 *
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