Assembled Nanodiscs Mouse/Rat


Nanodisc

Mouse/Rat assembled nanodiscs

Assembled nanodiscs can replace detergent in cell-free expression reactions. The nascent membrane protein integrates into the nanodiscs, forming a stable complex. As human scaffold proteins might interfere with certain applications such as immunization of mice or rats with human antigens, nanodiscs containing animal homologue proteins are introduced. Nanodiscs are conveniently aliquoted in 50 µL solutions of 0.5 mM concentration. End concentrations in the cell-free reaction typically range from 20-80 µM. As protein activity may depend on the lipid composition of the assembled nanodisc, it is recommended to test several lipids or lipid mixtures. Please note that assembled nanodiscs are intended for use in cell-free expression reactions only. For the reconstitution of membrane proteins already solubilized in detergent, please refer to lyophilized mouse membrane scaffold proteins.

Why mouse or rat assembled nanodiscs?

  • Ideal in combination with E.coE.coli cell-free expression lysates
  • No interference in mouse/rat immunization experiments
  • DMPC and POPC phospholipids available
  • MSP1D1 (wild-type) and MSPE3D1 scaffold proteins
  • Biotinylation and other lipid modifications optional

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 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 yellow-capped plastic vial containing the lyophilized MSP1D1 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 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 yellow-capped plastic vial containing the lyophilized MSP1D1 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

Mouse/Rat assembled nanodiscs

Assembled nanodiscs can replace detergent in cell-free expression reactions. The nascent membrane protein integrates into the nanodiscs, forming a stable complex. As human scaffold proteins might interfere with certain applications such as immunization of mice or rats with human antigens, nanodiscs containing animal homologue proteins are introduced. Nanodiscs are conveniently aliquoted in 50 µL solutions of 0.5 mM concentration. End concentrations in the cell-free reaction typically range from 20-80 µM. As protein activity may depend on the lipid composition of the assembled nanodisc, it is recommended to test several lipids or lipid mixtures. Please note that assembled nanodiscs are intended for use in cell-free expression reactions only. For the reconstitution of membrane proteins already solubilized in detergent, please refer to lyophilized mouse membrane scaffold proteins.

Why mouse or rat assembled nanodiscs?

  • Ideal in combination with E.coE.coli cell-free expression lysates
  • No interference in mouse/rat immunization experiments
  • DMPC and POPC phospholipids available
  • MSP1D1 (wild-type) and MSPE3D1 scaffold proteins
  • Biotinylation and other lipid modifications optional

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 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 yellow-capped plastic vial containing the lyophilized MSP1D1 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 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 yellow-capped plastic vial containing the lyophilized MSP1D1 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 Mouse/Rat
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Nanodisc mouse MSP1D1-His_DMPC (50 µL) Nanodisc mouse MSP1D1-His_DMPC (50 µL)
50 microliter pre-assembled nanodisc, 0.5 mM concentrated, containing mouse MSP1D1-His protein and DMPC. For use in cell-free reactions only. Shipped on dry ice - additional freight cost apply.
Article number: 26711
Sales price: €420.30 * €467.00 *
Nanodisc mouse MSP1D1-His_POPC (50 µL) Nanodisc mouse MSP1D1-His_POPC (50 µL)
50 microliter pre-assembled nanodisc, 0.5 mM concentrated, containing mouse MSP1D1-His protein and POPC. For use in cell-free reactions only. Shipped on dry ice - additional freight cost apply.
Article number: 26713
Sales price: €420.30 * €467.00 *
Nanodisc mouse MSP1D1-His_DMPG (50 µL) Nanodisc mouse MSP1D1-His_DMPG (50 µL)
50 microliter pre-assembled nanodisc, 0.5 mM concentrated, containing mouse MSP1D1-His protein and DMPG. For use in cell-free reactions only. Shipped on dry ice - additional freight cost apply.
Article number: 26715
Sales price: €420.30 * €467.00 *
Nanodisc mouse MSP1E3D1-His_DMPC (50 µL) Nanodisc mouse MSP1E3D1-His_DMPC (50 µL)
50 microliter pre-assembled nanodisc, 0.5 mM concentrated, containing mouse MSP1E3D1-His protein and DMPC. For use in cell-free reactions only. Shipped on dry ice - additional freight cost apply.
Article number: 26751
Sales price: €420.30 * €467.00 *
Nanodisc mouse MSP1E3D1-His_POPC (50 µL) Nanodisc mouse MSP1E3D1-His_POPC (50 µL)
50 microliter pre-assembled nanodisc, 0.5 mM concentrated, containing mouse MSP1E3D1-His protein and POPC. For use in cell-free reactions only. Shipped on dry ice - additional freight cost apply.
Article number: 26753
Sales price: €420.30 * €467.00 *
Nanodisc mouse MSP1E3D1-His_DMPG (50 µL) Nanodisc mouse MSP1E3D1-His_DMPG (50 µL)
50 microliter pre-assembled nanodisc, 0.5 mM concentrated, containing mouse MSP1E3D1-His protein and DMPG. For use in cell-free reactions only. Shipped on dry ice - additional freight cost apply.
Article number: 26755
Sales price: €420.30 * €467.00 *
Nanodisc rat MSP1D1-His_DMPC (50 µL) Nanodisc rat MSP1D1-His_DMPC (50 µL)
50 microliter pre-assembled nanodisc, 0.5 mM concentrated, containing rat MSP1D1-His protein and DMPC. For use in cell-free reactions only. Shipped on dry ice - additional freight cost apply.
Article number: 26911
Sales price: €420.30 * €467.00 *
Nanodisc rat MSP1D1-His_POPC (50 µL) Nanodisc rat MSP1D1-His_POPC (50 µL)
50 microliter pre-assembled nanodisc, 0.5 mM concentrated, containing rat MSP1D1-His protein and POPC. For use in cell-free reactions only. Shipped on dry ice - additional freight cost apply.
Article number: 26913
Sales price: €420.30 * €467.00 *
Nanodisc rat MSP1D1-His_DMPG (50 µL) Nanodisc rat MSP1D1-His_DMPG (50 µL)
50 microliter pre-assembled nanodisc, 0.5 mM concentrated, containing rat MSP1D1-His protein and DMPG. For use in cell-free reactions. Shipped on dry ice - additional freight cost may apply.
Article number: 26915
Sales price: €420.30 * €467.00 *
Nanodisc rat MSP1E3D1-His_DMPC (50 µL) Nanodisc rat MSP1E3D1-His_DMPC (50 µL)
50 microliter pre-assembled nanodisc, 0.5 mM concentrated, containing rat MSP1E3D1-His protein and DMPC. For use in cell-free reactions only. Shipped on dry ice - additional freight cost apply.
Article number: 26951
Sales price: €420.30 * €467.00 *
Nanodisc rat MSP1E3D1-His_POPC (50 µL) Nanodisc rat MSP1E3D1-His_POPC (50 µL)
50 microliter pre-assembled nanodisc, 0.5 mM concentrated, containing rat MSP1E3D1-His protein and POPC. For use in cell-free reactions. For use in cell-free reactions only. Shipped on dry ice - additional freight cost apply.
Article number: 26953
Sales price: €420.30 * €467.00 *
Nanodisc rat MSP1E3D1-His_DMPG (50 µL) Nanodisc rat MSP1E3D1-His_DMPG (50 µL)
50 microliter pre-assembled nanodisc, 0.5 mM concentrated, containing rat MSP1E3D1-His protein and POPC. For use in cell-free reactions. For use in cell-free reactions only. Shipped on dry ice - additional freight cost apply.
Article number: 26955
Sales price: €420.30 * €467.00 *
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