Nanodisc MSP untagged


Nanodisc

Nanodisc membrane scaffold proteins (MSPs) - untagged

Membrane scaffold proteins (MSPs) are typically used for the reconstitution of already isolated proteins. A variety of membrane scaffold proteins (MSPs) are available that can be assembled to nanodiscs of different sizes to accommodate proteins with varying numbers of transmembrane domains. If the protein of interest itself carries a his-tag, we recommend to use untagged MSP proteins for assembly. All our human nanodisc variants are now available as an untagged version. For use in cell-free expression reactions, pre-assembled nanodiscs are available, both as his-tagged and untagged variants.

Why Membrane scaffold proteins?

  • Variety of lyophilized membrane scaffold proteins available
  • Stable, lyophilized protein preparations in convenient aliquots
  • Can be combined with different phospholipids
  • Detailed protocols and expert support provided
 ProteinYearAuthor
Nanolipoprotein particles 2016 Wade Z., Kaitlin J., Darryl S., Subhash R., and Marjorie L.1
RyR1 and TcdA1 toxin subunit 2017 Efremov R., Gatsogiannis C., Raunser S.2
- 2018  Carvalho V., Pronk W., Engel, H.3
DmelOR10a, DmelOR22a, DmelOR35a, and DmelOR71a 2019 Murugathas T., Zheng H. Y., Colbert D., Kralicek A., Carraher C., Plank N.4
BSA 2019 Damiati S., Scheberl A., Zayni S., Damiati S., Schuster B., Kompella U.5
ETB 2016 Rues R.-B., Henrich E., Boland C., Caffrey M., Bernhard F.6
TcdA 2016 Gatsogiannis C., Merino F., Prumbaum D., Roderer D., Leidreiter F., Meusch D., Raunser S.7
hsc-70 2016 Morozova K., Clement C., Kaushik S., Stiller B., Arias E., Ahmad A., Rauch J., Chatterjee V., Melis C., Scharf B., Gestwicki J., Cuervo A., Zuiderweg E., Santambrogio L.8

 

Features

Purpose Forms nanodisc structures in combination with phospholipids
Specifity Stabilization of membrane proteins
Characteristics MSP1D1, MSP1E3D1 or MSP2N2 wild-type protein
Resulting nanodisc diameter: ca. 7-17 nm
Purity > 90 %
Component Lyophilized MSP protein
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: Reconstitution of membrane proteins into nanodisc using MSP1D1 protein and DMPC phospholipids
 
 
  1. Prepare a membrane protein solution containing your target protein of interest. As a starting point, use 200 µL of the protein solution at a concentration of 2-5 mg/mL.
  2. Determine the molar concentration of target protein solution, using the molecular weight and extinction coefficient e.g. at 280 nm.
  3. . Calculate the required amount of MSP protein to be 20 times the molar quantity of the target protein.
  4. Calculate the required amount of POPC to be 40 times the molar quantitiy of the MSP protein (or 800 times the molar quantity of the target protein).
  5. Resuspend the POPC in 500 µL ND Lipid Buffer II and incubate for 20 min at 37°C to fully solubilize the phospholipid.
  6. Resuspend the MSP protein in 500 µL ND Protein Buffer in a 2 mL microtube. Add the target protein, solubilized in detergent solution.
  7. Add the POPC solution to the MSP and membrane protein solution, and incubate the entire mix at 4°C for 2 h.
  8. During the incubation, equilibrate 2.5 g biobeads or similar adsorbant in ND Protein Buffer according to the manufacturer’s instructions. Degas the solution by ultrasound to remove any oxygen solubilized in the biobead solution.
  9. Add the protein/POPC mix to 750 µL of the biobead solution and incubate at 4°C on an end over end shaker for 8-12 h.
  10. . . Spin the solution at 10-12,000 x g for 2 min, and transfer the supernatant to a fresh tube. Add 750 µL of fresh equilibrated biobead solution and incubate at 4°C on an end over end shaker for 8-12 h.
  11. Repeat step 10 at least one more time to ensure complete detergent removal.
  12. Remove the supernatant, and filter the nanodisc mix through a 0.45 micron filter to remove precipitates that might have formed during incubation.
  13. . . Apply the nanodisc mix on a gel filtration column. Apply the mix in several portions if necessary. Monitor absorbance at 280 nm.
  14. . Collect fractions of ca. 500 µL size, and analyze the samples by SDS PAGE. MSP proteins have an apparent molecular mass of around 20 kDa.
  15. Concentrate the elution fractions which contain the nanodiscs with inserted membrane protein using protein concentrators.
  16. Freeze the nanodiscs containing membrane proteins at -80°C in a solution containing 10% glycerol for future use, or use them directly in the desired experiment.

 
   
B.POPC: Reconstitution of membrane proteins into nanodisc using MSP1D1 protein and POPC phospholipids
 
 
  1. Prepare a membrane protein solution containing your target protein of interest. As a starting point, use 200 μL of the protein solution at a concentration of 2-5 mg/mL. Note: Depending on the target protein, required protein concentrations might be up to 20 mg/mL.
  2. Determine the molar concentration of target protein solution, using the molecular weight and extinction coefficient e.g. at 280 nm.
  3. Calculate the required amount of MSP protein to be 20 times the molar quantity of the target protein.
  4. Calculate the required amount of POPC to be 55 times the molar quantitiy of the MSP protein (or 1,100 times the molar quantity of the target protein). Example: Target protein has a MW of 45,000 Da. Therefore, 500 μg protein in a 200 μL solution corresponds to 11 nmol of target protein in the 1 ml reaction mix. To obtain a 20 fold excess of MSP protein, use 220 nmol of MSP, i.e. 5.57 mg (MW 25.309 g/mol). For a 55 fold excess of phospholipid, use 12.1 μmol or 9.2 mg of POPC (MW 760 g/mol).
  5. Resuspend the POPC in 500 μL ND Lipid Buffer II and incubate for 20 min at 37°C to fully solubilize the phospholipid.
  6. Resuspend the MSP protein in 500 μL ND Protein Buffer in a 2 mL microtube. Add the target protein, solubilized in detergent solution. Note: The protein was lyophilized from a solution containing 4 mg/ml protein in 20 mM Tris pH 7.4, 100 mM NaCl, 0.5 mM EDTA.
  7. Add the POPC solution to the MSP and membrane protein solution, and incubate the entire mix at 4°C for 2 h.
  8. During the incubation, equilibrate 2.5 g biobeads or similar adsorbant in ND Protein Buffer according to the manufacturer's instructions. Degas the solution by ultrasound to remove any oxygen solubilized in the biobead solution.
  9. Add the protein/POPC mix to 750 μL of the biobead solution and incubate at 4°C on an end over end shaker for 8-12 h.
  10. Spin the solution at 10-12,000 x g for 2 min, and transfer the supernatant to a fresh tube. Add 750 μL of fresh equilibrated biobead solution and incubate at 4°C on an end over end shaker for 8-12 h.
  11. Repeat step 10 at least one more time to ensure complete detergent removal.
  12. Remove the supernatant, and filter the nanodisc mix through a 0.45 micron filter to remove precipitates that might have formed during incubation.
  13. Apply the nanodisc mix on a gel filtration column. Apply the mix in several portions if necessary. Monitor absorbance at 280 nm.
  14. Collect fractions of ca. 500 µL size, and analyze the samples by SDS PAGE. MSP proteins have an apparent molecular mass of around 20 kDa.
  15. Concentrate the elution fractions which contain the nanodiscs with inserted membrane protein using protein concentrators.
  16. Freeze the nanodiscs containing membrane proteins at -80°C in a solution containing 10% glycerol for future use, or use them directly in the desired experiment.

References

  1. Zeno, Wade F., et al. "Dynamics of crowding-induced mixing in phase separated lipid bilayers." The Journal of Physical Chemistry B 120.43 (2016): 11180-11190.
  2. Efremov, Rouslan G., Christos Gatsogiannis, and Stefan Raunser. "Lipid nanodiscs as a tool for high-resolution structure determination of membrane proteins by single-particle cryo-EM." Methods in enzymology. Vol. 594. Academic Press, 2017. 1-30.
  3. Carvalho, Vanessa, Joachim W. Pronk, and Andreas H. Engel. "Characterization of Membrane Proteins Using Cryo‐Electron Microscopy." Current protocols in protein science 94.1 (2018): e72.
  4. Murugathas, Thanihaichelvan, et al. "Biosensing with Insect Odorant Receptor Nanodiscs and Carbon Nanotube Field-Effect Transistors." ACS applied materials & interfaces 11.9 (2019): 9530-9538.
  5. Damiati, Samar, et al. "Albumin-bound nanodiscs as delivery vehicle candidates: Development and characterization." Biophysical chemistry 251 (2019): 106178.
  6. Rues, Ralf-Bernhardt, et al. "Cell-free production of membrane proteins in Escherichia coli lysates for functional and structural studies." Heterologous Expression of Membrane Proteins. Humana Press, New York, NY, 2016. 1-21.
  7. Gatsogiannis, Christos, et al. "Membrane insertion of a Tc toxin in near-atomic detail." Nature structural & molecular biology 23.10 (2016): 884.
  8. Morozova, Kateryna, et al. "Structural and biological interaction of hsc-70 protein with phosphatidylserine in endosomal microautophagy." Journal of Biological Chemistry 291.35 (2016): 18096-18106.
Nanodisc

Nanodisc membrane scaffold proteins (MSPs) - untagged

Membrane scaffold proteins (MSPs) are typically used for the reconstitution of already isolated proteins. A variety of membrane scaffold proteins (MSPs) are available that can be assembled to nanodiscs of different sizes to accommodate proteins with varying numbers of transmembrane domains. If the protein of interest itself carries a his-tag, we recommend to use untagged MSP proteins for assembly. All our human nanodisc variants are now available as an untagged version. For use in cell-free expression reactions, pre-assembled nanodiscs are available, both as his-tagged and untagged variants.

Why Membrane scaffold proteins?

  • Variety of lyophilized membrane scaffold proteins available
  • Stable, lyophilized protein preparations in convenient aliquots
  • Can be combined with different phospholipids
  • Detailed protocols and expert support provided
 ProteinYearAuthor
Nanolipoprotein particles 2016 Wade Z., Kaitlin J., Darryl S., Subhash R., and Marjorie L.1
RyR1 and TcdA1 toxin subunit 2017 Efremov R., Gatsogiannis C., Raunser S.2
- 2018  Carvalho V., Pronk W., Engel, H.3
DmelOR10a, DmelOR22a, DmelOR35a, and DmelOR71a 2019 Murugathas T., Zheng H. Y., Colbert D., Kralicek A., Carraher C., Plank N.4
BSA 2019 Damiati S., Scheberl A., Zayni S., Damiati S., Schuster B., Kompella U.5
ETB 2016 Rues R.-B., Henrich E., Boland C., Caffrey M., Bernhard F.6
TcdA 2016 Gatsogiannis C., Merino F., Prumbaum D., Roderer D., Leidreiter F., Meusch D., Raunser S.7
hsc-70 2016 Morozova K., Clement C., Kaushik S., Stiller B., Arias E., Ahmad A., Rauch J., Chatterjee V., Melis C., Scharf B., Gestwicki J., Cuervo A., Zuiderweg E., Santambrogio L.8

 

Features

Purpose Forms nanodisc structures in combination with phospholipids
Specifity Stabilization of membrane proteins
Characteristics MSP1D1, MSP1E3D1 or MSP2N2 wild-type protein
Resulting nanodisc diameter: ca. 7-17 nm
Purity > 90 %
Component Lyophilized MSP protein
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: Reconstitution of membrane proteins into nanodisc using MSP1D1 protein and DMPC phospholipids
 
 
  1. Prepare a membrane protein solution containing your target protein of interest. As a starting point, use 200 µL of the protein solution at a concentration of 2-5 mg/mL.
  2. Determine the molar concentration of target protein solution, using the molecular weight and extinction coefficient e.g. at 280 nm.
  3. . Calculate the required amount of MSP protein to be 20 times the molar quantity of the target protein.
  4. Calculate the required amount of POPC to be 40 times the molar quantitiy of the MSP protein (or 800 times the molar quantity of the target protein).
  5. Resuspend the POPC in 500 µL ND Lipid Buffer II and incubate for 20 min at 37°C to fully solubilize the phospholipid.
  6. Resuspend the MSP protein in 500 µL ND Protein Buffer in a 2 mL microtube. Add the target protein, solubilized in detergent solution.
  7. Add the POPC solution to the MSP and membrane protein solution, and incubate the entire mix at 4°C for 2 h.
  8. During the incubation, equilibrate 2.5 g biobeads or similar adsorbant in ND Protein Buffer according to the manufacturer’s instructions. Degas the solution by ultrasound to remove any oxygen solubilized in the biobead solution.
  9. Add the protein/POPC mix to 750 µL of the biobead solution and incubate at 4°C on an end over end shaker for 8-12 h.
  10. . . Spin the solution at 10-12,000 x g for 2 min, and transfer the supernatant to a fresh tube. Add 750 µL of fresh equilibrated biobead solution and incubate at 4°C on an end over end shaker for 8-12 h.
  11. Repeat step 10 at least one more time to ensure complete detergent removal.
  12. Remove the supernatant, and filter the nanodisc mix through a 0.45 micron filter to remove precipitates that might have formed during incubation.
  13. . . Apply the nanodisc mix on a gel filtration column. Apply the mix in several portions if necessary. Monitor absorbance at 280 nm.
  14. . Collect fractions of ca. 500 µL size, and analyze the samples by SDS PAGE. MSP proteins have an apparent molecular mass of around 20 kDa.
  15. Concentrate the elution fractions which contain the nanodiscs with inserted membrane protein using protein concentrators.
  16. Freeze the nanodiscs containing membrane proteins at -80°C in a solution containing 10% glycerol for future use, or use them directly in the desired experiment.

 
   
B.POPC: Reconstitution of membrane proteins into nanodisc using MSP1D1 protein and POPC phospholipids
 
 
  1. Prepare a membrane protein solution containing your target protein of interest. As a starting point, use 200 μL of the protein solution at a concentration of 2-5 mg/mL. Note: Depending on the target protein, required protein concentrations might be up to 20 mg/mL.
  2. Determine the molar concentration of target protein solution, using the molecular weight and extinction coefficient e.g. at 280 nm.
  3. Calculate the required amount of MSP protein to be 20 times the molar quantity of the target protein.
  4. Calculate the required amount of POPC to be 55 times the molar quantitiy of the MSP protein (or 1,100 times the molar quantity of the target protein). Example: Target protein has a MW of 45,000 Da. Therefore, 500 μg protein in a 200 μL solution corresponds to 11 nmol of target protein in the 1 ml reaction mix. To obtain a 20 fold excess of MSP protein, use 220 nmol of MSP, i.e. 5.57 mg (MW 25.309 g/mol). For a 55 fold excess of phospholipid, use 12.1 μmol or 9.2 mg of POPC (MW 760 g/mol).
  5. Resuspend the POPC in 500 μL ND Lipid Buffer II and incubate for 20 min at 37°C to fully solubilize the phospholipid.
  6. Resuspend the MSP protein in 500 μL ND Protein Buffer in a 2 mL microtube. Add the target protein, solubilized in detergent solution. Note: The protein was lyophilized from a solution containing 4 mg/ml protein in 20 mM Tris pH 7.4, 100 mM NaCl, 0.5 mM EDTA.
  7. Add the POPC solution to the MSP and membrane protein solution, and incubate the entire mix at 4°C for 2 h.
  8. During the incubation, equilibrate 2.5 g biobeads or similar adsorbant in ND Protein Buffer according to the manufacturer's instructions. Degas the solution by ultrasound to remove any oxygen solubilized in the biobead solution.
  9. Add the protein/POPC mix to 750 μL of the biobead solution and incubate at 4°C on an end over end shaker for 8-12 h.
  10. Spin the solution at 10-12,000 x g for 2 min, and transfer the supernatant to a fresh tube. Add 750 μL of fresh equilibrated biobead solution and incubate at 4°C on an end over end shaker for 8-12 h.
  11. Repeat step 10 at least one more time to ensure complete detergent removal.
  12. Remove the supernatant, and filter the nanodisc mix through a 0.45 micron filter to remove precipitates that might have formed during incubation.
  13. Apply the nanodisc mix on a gel filtration column. Apply the mix in several portions if necessary. Monitor absorbance at 280 nm.
  14. Collect fractions of ca. 500 µL size, and analyze the samples by SDS PAGE. MSP proteins have an apparent molecular mass of around 20 kDa.
  15. Concentrate the elution fractions which contain the nanodiscs with inserted membrane protein using protein concentrators.
  16. Freeze the nanodiscs containing membrane proteins at -80°C in a solution containing 10% glycerol for future use, or use them directly in the desired experiment.

References

  1. Zeno, Wade F., et al. "Dynamics of crowding-induced mixing in phase separated lipid bilayers." The Journal of Physical Chemistry B 120.43 (2016): 11180-11190.
  2. Efremov, Rouslan G., Christos Gatsogiannis, and Stefan Raunser. "Lipid nanodiscs as a tool for high-resolution structure determination of membrane proteins by single-particle cryo-EM." Methods in enzymology. Vol. 594. Academic Press, 2017. 1-30.
  3. Carvalho, Vanessa, Joachim W. Pronk, and Andreas H. Engel. "Characterization of Membrane Proteins Using Cryo‐Electron Microscopy." Current protocols in protein science 94.1 (2018): e72.
  4. Murugathas, Thanihaichelvan, et al. "Biosensing with Insect Odorant Receptor Nanodiscs and Carbon Nanotube Field-Effect Transistors." ACS applied materials & interfaces 11.9 (2019): 9530-9538.
  5. Damiati, Samar, et al. "Albumin-bound nanodiscs as delivery vehicle candidates: Development and characterization." Biophysical chemistry 251 (2019): 106178.
  6. Rues, Ralf-Bernhardt, et al. "Cell-free production of membrane proteins in Escherichia coli lysates for functional and structural studies." Heterologous Expression of Membrane Proteins. Humana Press, New York, NY, 2016. 1-21.
  7. Gatsogiannis, Christos, et al. "Membrane insertion of a Tc toxin in near-atomic detail." Nature structural & molecular biology 23.10 (2016): 884.
  8. Morozova, Kateryna, et al. "Structural and biological interaction of hsc-70 protein with phosphatidylserine in endosomal microautophagy." Journal of Biological Chemistry 291.35 (2016): 18096-18106.
Nanodisc MSP untagged
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MSP2N2, lyophilized protein MSP2N2, lyophilized protein
Membrane scaffold protein (MSP) 2N2, His tag removed by proteolytic digest. 2 mg lyophilized protein
Article number: 26182
Sales price: From €296.00 *
MSP1D1, lyophilized protein MSP1D1, lyophilized protein
Membrane scaffold protein (MSP)1D1, His tag removed by proteolytic digest. 2 mg lyophilized protein
Article number: 26132
Sales price: From €238.00 *
MSP1D1 dH5, lyophilized protein MSP1D1 dH5, lyophilized protein
Membrane scaffold protein MSP1D1ΔH5 with N-terminal His-tag, 2 mg lyophilized protein ► for nanodisc diameters of 7-8 nm
Article number: 26142
Sales price: From €238.00 *
MSP1E3D1, lyophilized protein MSP1E3D1, lyophilized protein
Membrane scaffold protein 1 E3D1, His-tag removed by protease digest. 2 mg lyophilized protein
Article number: 26162
Sales price: From €238.00 *
mouse MSP1D1, lyophilized protein mouse MSP1D1, lyophilized protein
(Mouse) Membrane scaffolding protein MSP1D1 for nanodisc assembly. For scaffolding diameters of 9-10 nanometers. Lyophilized.
Article number: 26532
Sales price: From €259.00 *
mouse MSP1E3D1, lyophilized protein mouse MSP1E3D1, lyophilized protein
Membrane scaffolding protein MSP1E3D1 for nanodisc assembly. Creates scaffolding diameters of 12-14 nanometers. Lyophilized.
Article number: 26562
Sales price: From €259.00 *
mouse MSP2N2, lyophilized protein mouse MSP2N2, lyophilized protein
(Mouse) Membrane scaffolding protein MSP2N2 for nanodisc assembly. For scaffolding diameters of 17 nanometers. Lyophilized.
Article number: 26582
Sales price: From €333.00 *
MSP1D1 dH5-D73C, lyophilized protein MSP1D1 dH5-D73C, lyophilized protein
The new MSP1D1 dH5-D73C-His nanodisc from Cube Biotech contains the amino acid cystein.
Article number: 27242
Sales price: From €155.00 *
MSP1E3D1-D73C, lyophilized protein MSP1E3D1-D73C, lyophilized protein
The new MSP1E3D1-D73C-His nanodisc from Cube Biotech contains the amino acid cystein.
Article number: 27262
Sales price: From €155.00 *
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