Co-NTA MagBeads


MagBeads
The His tag is the most widely used affinity tag due to its small size and versatility under native and denaturing conditions, as well as in presence of detergents and many other additives. Magnetic beads are ideal for protein purification from dilute supernatants and for pull-down experiments. The agarose surface of our MagBeads is identical to that of PureCube Agarose, making them an ideal combination for small-scale screening and upscale reactions.In addition to the widely used Ni-NTA MagBeads, Cube Biotech offers high-performance PureCube Co-NTA MagBeads for purification of his-tagged proteins. Specificity of this transition metal for histidine stretches is typically higher than that of nickel (see Fig. 1). PureCube Co-NTA MagBeads are ferrimagnetic agarose beads coupled to an NTA chelating ligand, loaded with cobalt ions. PureCube Co-NTA MagBeads are delivered as a 25% suspension.

The Cobalt-NTA MagBeads provide:


Cobalt Beads in detail:

Affinity metal Ions
Fig. 1: Affinity and specificity of metal ions commonly used for IMAC. Loading an IMAC resin with different metal ions can adjust the affinity and specificity to optimize the purity and yield of a purified protein.
 

Different metal ions confer different binding affinity and specificity

Loading different metal ions to a resin results in differing affinity and specificity for a his-tagged protein (Fig.1). Generally, cobalt exhibits the higest binding specificity of commonly used IMAC metal ions, leading to relatively low yields but high purity. Copper, at the other end of the spectrum, has a high affinity leading to high yields but unspecific binding. In searching for the optimal resin to purify a protein, it is recommended to explore different chelating ligands (IDA or NTA) and different metal ions.
Ni-NTA MagBeads
Fig.2: Highest yield and specificity obtained with PureCube Co-NTA MagBeads. SDS-PAGE of radiolabeled, his-tagged proteins purified from an in vitro transcription/translation reaction with PureCube NTA MagBeads and comparable products from other suppliers.Rxn: total reaction, T Co: Talon Cobalt MagBeads 10 µl 5%, C Ni: PureCube Co-NTA MagBeads, 5 µl 5%; C Co: PureCube Co-NTA MagBeads, 5 µl 5%; 5 Ni: Co-NTA MagBeads from Supplier 5, 10 µl 5% suspension. Note that only half the amount of PureCube Ni and Co-NTA MagBeads were employed in each reaction compared to competitor beads. Data kindly provided by Dr. Daniel Kraut, Department of Chemistry, Villanova University, Pennsylvania, USA
 

Nickel vs Cobalt IMAC

Depending on applications, nickel or cobalt IMAC may be the purification matrix of choice. For example, in customer data shown in Fig. 2, radio-labeled, his-tagged proteins generated in an in vitro transcription/translation reaction were purified using Ni and Co IMAC magnetic beads from different suppliers. PureCube Co-NTA Magbeads showed highest binding capacity and highest purity of the his-tagged protein, which was then used as a substrate for proteasome studies.

Features

Usage Specific binding and purification of 6x his-tagged proteins
Specifity Affinity to His-tagged proteins
Binding capacity >30 mg/mL
Chelator stability Stable in buffer containing 10 mM DTT and 1 mM EDTA
Filling quantity Delivered as a 25 % suspension
Bead size 30 μm
Bead Ligand Co-NTA
Required equipment
 
  • Lysis Buffer
  • Wash Buffer
  • Elution Buffer
  • Ice bath
  • Refrigerated centrifuge for 50 mL tube (min 10,000 x g)
  • 50 mL centrifuge tube
  • Micropipettor and Micropipetting tips
  • Disposable gravity flow columns with capped bottom outlet, 2 ml
  •  pH meter
  • End-over-end shaker
  • SDS-PAGE buffers, reagents and equipment Optional: Western Blot reagents and equipment

Applications

All protocols are also avaible as PDF-Files on the Protocols & Datasheets page.
   
A.Protocol for purification under native conditions:
 
 
  1. Thaw the E. coli cell pellet on ice. Optional: Freezing the cell pellet at -20 °C for 30 min prior to incubation at room temperature improves lysis by lysozyme.
  2. Resuspend the cell pellet in 1 mL Native Lysis Buffer supplemented with 1 mg/mL lysozyme.
  3. Add 6 U Benzonase® (3 units/mL bacterial culture) to the lysate to reduce viscosity caused by genomic DNA.
  4. Incubate for 30 min on ice, if necessary. Otherwise, incubating at room temperature (20-25 °C) may be more efficient.
  5. Centrifuge the lysate for 30 min at 10,000xg and 4 °C. Collect the supernatant. Note: The supernatant contains the cleared lysate fraction. We recommend to take aliquots of all fractions for SDS-PAGE analysis.
  6. Resuspend the PureCube Co-NTA MagBeads by vortexing. Transfer 40 μL of the 25 % magnetic bead suspension into a conical microcentrifuge tube. Note: Depending on the protein expression rate, the quantity of magnetic bead suspension can be adjusted from 2-200 μL.
  7. Add 500 μL Native Lysis Buffer and mix by vortexing. Place the tube on a magnetic microtube stand until the beads are separated and discard the supernatant.
  8. Pipet 1 mL of the cleared lysate onto the equilibrated magnetic beads, and incubate the lysate-magnetic bead mixture at 4 °C for 1 h on an end-over-end shaker.
  9. Place the tube on the magnetic microtube stand until the beads separate and remove the supernatant. Tip: Briefly centrifuge the sample before placing it on the magnetic separator in order to collect liquid from the lid.
  10. Remove the tube from the magnet. Add 500 μL Native Wash Buffer and mix by vortexing. Place the tube again on the magnetic microtube stand and allow the beads to separate. Remove the supernatant.
  11. Repeat step 10 twice.
  12. Elute the His-tagged protein using 100 μL Native Elution Buffer. Note: Depending on the protein expression rate and desired protein concentration, the elution volume can be adjusted from 25 to 500 μL..
  13. Repeat step 12 three times. Collect each elution fraction in a separate tube and determine the protein concentration of each fraction.
  14. Analyze all fractions by SDS-PAGE. Note: Do not boil membrane proteins. Instead, incubate the sample at 46 °C for 30 min in preparation for SDS-PAGE analysis.
  15. Optional: Perform Western Blot experiment using PentaHis Antibody.
 B.Protocol for purification under denaturing conditions:
 
 
  1. Thaw the E. coli cell pellet on ice.
  2. Resuspend the cell pellet in 1 mL Denaturing Lysis Buffer. Optional: Benzonase® can be added to the lysate to reduce viscosity caused by nucleic acids (3 U/mL bacterial culture). In addition, nucleic acids can be sheared by passing the lysate 10 times through a fine-gauge needle.
  3. Incubate at room temperature for 30 min on an end-over-end shaker.
  4. Centrifuge the lysate for 30 min at room temperature and 10,000 x g. Collect the supernatant. Note: The supernatant contains the cleared lysate fraction. We recommend to take aliquots of all fractions for SDS-PAGE analysis.
  5. Pipet 1 mL of the cleared lysate into a conical microcentrifuge tube.
  6. Resuspend the PureCube Co-NTA MagBeads by vortexing. Transfer 40 μL of the 25% magnetic beads suspension onto the lysate. Note: Depending on the protein expression rate and desired protein concentration, the elution volume can be adjusted from 25 to 500 µl.
  7. Incubate the lysate-magnetic bead mixture at room temperature for 1 h on an end-over-end shaker..
  8. Place the tube on the magnetic microtube stand until the beads separate and remove the supernatant. Tip: Briefly centrifuge the sample before placing it on the magnetic separator in order to collect liquid from the lid.
  9. Remove the tube from the magnet. Add 500 µL Denaturing Wash Buffer and mix by vortexing. Place the tube again on the magnetic microtube stand and allow the beads to separate. Remove the supernatant.
  10. Repeat step 9 twice.
  11. Elute the His-tagged protein using 100 μL Denaturing Elution Buffer. Note: Depending on the protein expression rate and desired protein concentration, the elution volume can be adjusted from 25 to 500 µl..
  12. Repeat step 11. Collect each elution fraction in a separate tube and determine the protein concentration of each fraction.
  13. Analyze all fractions by SDS-PAGE. Note: Do not boil membrane proteins. Instead, incubate samples at 46˚C for 30 min in preparation for SDS-PAGE analysis.
  14. Optional: Perform Western Blot experiment using PentaHis Antibody. If the target protein is acid-labile, elution can be performed with 250-500 mM imidazole.
 C.Wash protocol - Recommended after each use
 
 
Note: "bv" refers to column bed volume, i.e., 10 bv calls for 10 mL of buffer for a 1 mL column bed volume.
  1. Remove the majority of the fluid in the column containing the Co-NTA matrix. Add 10 bv dd water and allow the majority of the water volume to drip out of the column. Note: You can allow the fluid to drip through the column by gravity, or use a pressure bulb to gently force the fluid through the matrix. Ensure not to dry out the matrix.
  2. . Add 10 bv Wash Buffer to the column and allow the volume to completely flow through the matrix.
  3. Rinse the column again with 10 bv dd water.
  4. Add 10 bv 20% (v/v) ethanol and allow the majority of the volume to drip out of the column. The matrix is now ready to be re-used.
 D.Wash & Regeneration protocol without the use of DTT (recommended after each run, latest after 5 runs)
 
 
Note: "bv" refers to column bed volume, i.e., 10 bv calls for 10 mL of buffer for a 1 mL column bed volume.
  1. Remove the majority of the fluid in the column containing the Co-NTA matrix. Add 10 bv dd water and allow the majority of the volume to drip out of the column.
  2. Note: For removal of contaminations with very hydrophobic proteins or lipids, or precipitated proteins, incubate the matrix with one of the following chemicals for 1-2 h: 100% methanol, 100% ethanol, 8 M urea, 6 M guanidinium hydrochloride, 30% acetonitrile, or 1 M NaOH. Thoroughly wash with distilled water.
  3. Add 10 bv 100 mM EDTA to the column and allow the entire volume to flow through the matrix.
  4. Rinse the column again with 10 bv dd water
  5. Add 10 bv Wash Buffer to the column and allow the entire volume to flow through the matrix.
  6. Rinse the column with 10 bv dd water
  7. Add 10 bv 10m M CoSO4 to recharge the matrix. Allow the volume to drip through the column by gravity
  8. Rinse the column with 5 bv dd water.
  9. Wash twice with 5 bv dd water each.
  10. Wash with 5 bv 20 mM Tris pH 8.0.
  11. Wash twice with 5 bv dd water each.
  12. Add 10 bv of 20% (v/v) ethanol and allow the majority of the volume to drip out of the column. The matrix is now ready to be re-used.
 E.Wash & Regeneration protocol after the use of DTT (Neccessary after each run!)
 
 
Note: "bv" refers to column bed volume, i.e., 10 bv calls for 10 mL of buffer for a 1 mL column bed volume.
  1. Remove the majority of the fluid in the column containing the Co-NTA MagBeads. Add 10 bv dd water and allow the majority of the volume to drip out of the column.
  2. Briefly wash the resin with 10 bv 1–3% (v/v) HCl. Minimize the exposure time of the resin to HCl. Note: The concentration of HCl depends on the extent to which the resin is reduced. For example, 1% HCl was sufficient to strip Co-NTA MagBeads exposed to 1 mM DTT, 2% HCl for 5 mM DTT, and 3% for 10 mM DTT.
  3. Rinse the column with 10 bv dd water
  4. If the resin is not completely white, repeat steps 2 and 3. Otherwise, continue to step 4.
  5. Add 10 bv Wash Buffer and allow the majority of the volume to drip out of the column.
  6. Rinse the column with 10 bv dd water.
  7. Add 10 bv 10 mM CoSO4 to recharge the resin. Allow the volume to drip through the column by gravity.
  8. Rinse the column with 5 bv dd water
  9. Add 5 bv of Regeneration Buffer and incubate the matrix for 15 min at room temperature.
  10. Wash twice with 5 bv dd water each.
  11. Wash with 5 bv 20 mM Tris pH 8.0. Note: The extensive wash steps remove free nickel ions from the column, enhancing performance of the material in subsequent purifications, especially in presence of DTT.
  12. Wash twice with 5 bv dd water each
  13. . Add 10 bv 20% (v/v) ethanol and allow the majority of the volume to drip out of the column. The matrix is now ready to be re-used.
MagBeads
The His tag is the most widely used affinity tag due to its small size and versatility under native and denaturing conditions, as well as in presence of detergents and many other additives. Magnetic beads are ideal for protein purification from dilute supernatants and for pull-down experiments. The agarose surface of our MagBeads is identical to that of PureCube Agarose, making them an ideal combination for small-scale screening and upscale reactions.In addition to the widely used Ni-NTA MagBeads, Cube Biotech offers high-performance PureCube Co-NTA MagBeads for purification of his-tagged proteins. Specificity of this transition metal for histidine stretches is typically higher than that of nickel (see Fig. 1). PureCube Co-NTA MagBeads are ferrimagnetic agarose beads coupled to an NTA chelating ligand, loaded with cobalt ions. PureCube Co-NTA MagBeads are delivered as a 25% suspension.

The Cobalt-NTA MagBeads provide:


Cobalt Beads in detail:

Affinity metal Ions
Fig. 1: Affinity and specificity of metal ions commonly used for IMAC. Loading an IMAC resin with different metal ions can adjust the affinity and specificity to optimize the purity and yield of a purified protein.
 

Different metal ions confer different binding affinity and specificity

Loading different metal ions to a resin results in differing affinity and specificity for a his-tagged protein (Fig.1). Generally, cobalt exhibits the higest binding specificity of commonly used IMAC metal ions, leading to relatively low yields but high purity. Copper, at the other end of the spectrum, has a high affinity leading to high yields but unspecific binding. In searching for the optimal resin to purify a protein, it is recommended to explore different chelating ligands (IDA or NTA) and different metal ions.
Ni-NTA MagBeads
Fig.2: Highest yield and specificity obtained with PureCube Co-NTA MagBeads. SDS-PAGE of radiolabeled, his-tagged proteins purified from an in vitro transcription/translation reaction with PureCube NTA MagBeads and comparable products from other suppliers.Rxn: total reaction, T Co: Talon Cobalt MagBeads 10 µl 5%, C Ni: PureCube Co-NTA MagBeads, 5 µl 5%; C Co: PureCube Co-NTA MagBeads, 5 µl 5%; 5 Ni: Co-NTA MagBeads from Supplier 5, 10 µl 5% suspension. Note that only half the amount of PureCube Ni and Co-NTA MagBeads were employed in each reaction compared to competitor beads. Data kindly provided by Dr. Daniel Kraut, Department of Chemistry, Villanova University, Pennsylvania, USA
 

Nickel vs Cobalt IMAC

Depending on applications, nickel or cobalt IMAC may be the purification matrix of choice. For example, in customer data shown in Fig. 2, radio-labeled, his-tagged proteins generated in an in vitro transcription/translation reaction were purified using Ni and Co IMAC magnetic beads from different suppliers. PureCube Co-NTA Magbeads showed highest binding capacity and highest purity of the his-tagged protein, which was then used as a substrate for proteasome studies.

Features

Usage Specific binding and purification of 6x his-tagged proteins
Specifity Affinity to His-tagged proteins
Binding capacity >30 mg/mL
Chelator stability Stable in buffer containing 10 mM DTT and 1 mM EDTA
Filling quantity Delivered as a 25 % suspension
Bead size 30 μm
Bead Ligand Co-NTA
Required equipment
 
  • Lysis Buffer
  • Wash Buffer
  • Elution Buffer
  • Ice bath
  • Refrigerated centrifuge for 50 mL tube (min 10,000 x g)
  • 50 mL centrifuge tube
  • Micropipettor and Micropipetting tips
  • Disposable gravity flow columns with capped bottom outlet, 2 ml
  •  pH meter
  • End-over-end shaker
  • SDS-PAGE buffers, reagents and equipment Optional: Western Blot reagents and equipment

Applications

All protocols are also avaible as PDF-Files on the Protocols & Datasheets page.
   
A.Protocol for purification under native conditions:
 
 
  1. Thaw the E. coli cell pellet on ice. Optional: Freezing the cell pellet at -20 °C for 30 min prior to incubation at room temperature improves lysis by lysozyme.
  2. Resuspend the cell pellet in 1 mL Native Lysis Buffer supplemented with 1 mg/mL lysozyme.
  3. Add 6 U Benzonase® (3 units/mL bacterial culture) to the lysate to reduce viscosity caused by genomic DNA.
  4. Incubate for 30 min on ice, if necessary. Otherwise, incubating at room temperature (20-25 °C) may be more efficient.
  5. Centrifuge the lysate for 30 min at 10,000xg and 4 °C. Collect the supernatant. Note: The supernatant contains the cleared lysate fraction. We recommend to take aliquots of all fractions for SDS-PAGE analysis.
  6. Resuspend the PureCube Co-NTA MagBeads by vortexing. Transfer 40 μL of the 25 % magnetic bead suspension into a conical microcentrifuge tube. Note: Depending on the protein expression rate, the quantity of magnetic bead suspension can be adjusted from 2-200 μL.
  7. Add 500 μL Native Lysis Buffer and mix by vortexing. Place the tube on a magnetic microtube stand until the beads are separated and discard the supernatant.
  8. Pipet 1 mL of the cleared lysate onto the equilibrated magnetic beads, and incubate the lysate-magnetic bead mixture at 4 °C for 1 h on an end-over-end shaker.
  9. Place the tube on the magnetic microtube stand until the beads separate and remove the supernatant. Tip: Briefly centrifuge the sample before placing it on the magnetic separator in order to collect liquid from the lid.
  10. Remove the tube from the magnet. Add 500 μL Native Wash Buffer and mix by vortexing. Place the tube again on the magnetic microtube stand and allow the beads to separate. Remove the supernatant.
  11. Repeat step 10 twice.
  12. Elute the His-tagged protein using 100 μL Native Elution Buffer. Note: Depending on the protein expression rate and desired protein concentration, the elution volume can be adjusted from 25 to 500 μL..
  13. Repeat step 12 three times. Collect each elution fraction in a separate tube and determine the protein concentration of each fraction.
  14. Analyze all fractions by SDS-PAGE. Note: Do not boil membrane proteins. Instead, incubate the sample at 46 °C for 30 min in preparation for SDS-PAGE analysis.
  15. Optional: Perform Western Blot experiment using PentaHis Antibody.
 B.Protocol for purification under denaturing conditions:
 
 
  1. Thaw the E. coli cell pellet on ice.
  2. Resuspend the cell pellet in 1 mL Denaturing Lysis Buffer. Optional: Benzonase® can be added to the lysate to reduce viscosity caused by nucleic acids (3 U/mL bacterial culture). In addition, nucleic acids can be sheared by passing the lysate 10 times through a fine-gauge needle.
  3. Incubate at room temperature for 30 min on an end-over-end shaker.
  4. Centrifuge the lysate for 30 min at room temperature and 10,000 x g. Collect the supernatant. Note: The supernatant contains the cleared lysate fraction. We recommend to take aliquots of all fractions for SDS-PAGE analysis.
  5. Pipet 1 mL of the cleared lysate into a conical microcentrifuge tube.
  6. Resuspend the PureCube Co-NTA MagBeads by vortexing. Transfer 40 μL of the 25% magnetic beads suspension onto the lysate. Note: Depending on the protein expression rate and desired protein concentration, the elution volume can be adjusted from 25 to 500 µl.
  7. Incubate the lysate-magnetic bead mixture at room temperature for 1 h on an end-over-end shaker..
  8. Place the tube on the magnetic microtube stand until the beads separate and remove the supernatant. Tip: Briefly centrifuge the sample before placing it on the magnetic separator in order to collect liquid from the lid.
  9. Remove the tube from the magnet. Add 500 µL Denaturing Wash Buffer and mix by vortexing. Place the tube again on the magnetic microtube stand and allow the beads to separate. Remove the supernatant.
  10. Repeat step 9 twice.
  11. Elute the His-tagged protein using 100 μL Denaturing Elution Buffer. Note: Depending on the protein expression rate and desired protein concentration, the elution volume can be adjusted from 25 to 500 µl..
  12. Repeat step 11. Collect each elution fraction in a separate tube and determine the protein concentration of each fraction.
  13. Analyze all fractions by SDS-PAGE. Note: Do not boil membrane proteins. Instead, incubate samples at 46˚C for 30 min in preparation for SDS-PAGE analysis.
  14. Optional: Perform Western Blot experiment using PentaHis Antibody. If the target protein is acid-labile, elution can be performed with 250-500 mM imidazole.
 C.Wash protocol - Recommended after each use
 
 
Note: "bv" refers to column bed volume, i.e., 10 bv calls for 10 mL of buffer for a 1 mL column bed volume.
  1. Remove the majority of the fluid in the column containing the Co-NTA matrix. Add 10 bv dd water and allow the majority of the water volume to drip out of the column. Note: You can allow the fluid to drip through the column by gravity, or use a pressure bulb to gently force the fluid through the matrix. Ensure not to dry out the matrix.
  2. . Add 10 bv Wash Buffer to the column and allow the volume to completely flow through the matrix.
  3. Rinse the column again with 10 bv dd water.
  4. Add 10 bv 20% (v/v) ethanol and allow the majority of the volume to drip out of the column. The matrix is now ready to be re-used.
 D.Wash & Regeneration protocol without the use of DTT (recommended after each run, latest after 5 runs)
 
 
Note: "bv" refers to column bed volume, i.e., 10 bv calls for 10 mL of buffer for a 1 mL column bed volume.
  1. Remove the majority of the fluid in the column containing the Co-NTA matrix. Add 10 bv dd water and allow the majority of the volume to drip out of the column.
  2. Note: For removal of contaminations with very hydrophobic proteins or lipids, or precipitated proteins, incubate the matrix with one of the following chemicals for 1-2 h: 100% methanol, 100% ethanol, 8 M urea, 6 M guanidinium hydrochloride, 30% acetonitrile, or 1 M NaOH. Thoroughly wash with distilled water.
  3. Add 10 bv 100 mM EDTA to the column and allow the entire volume to flow through the matrix.
  4. Rinse the column again with 10 bv dd water
  5. Add 10 bv Wash Buffer to the column and allow the entire volume to flow through the matrix.
  6. Rinse the column with 10 bv dd water
  7. Add 10 bv 10m M CoSO4 to recharge the matrix. Allow the volume to drip through the column by gravity
  8. Rinse the column with 5 bv dd water.
  9. Wash twice with 5 bv dd water each.
  10. Wash with 5 bv 20 mM Tris pH 8.0.
  11. Wash twice with 5 bv dd water each.
  12. Add 10 bv of 20% (v/v) ethanol and allow the majority of the volume to drip out of the column. The matrix is now ready to be re-used.
 E.Wash & Regeneration protocol after the use of DTT (Neccessary after each run!)
 
 
Note: "bv" refers to column bed volume, i.e., 10 bv calls for 10 mL of buffer for a 1 mL column bed volume.
  1. Remove the majority of the fluid in the column containing the Co-NTA MagBeads. Add 10 bv dd water and allow the majority of the volume to drip out of the column.
  2. Briefly wash the resin with 10 bv 1–3% (v/v) HCl. Minimize the exposure time of the resin to HCl. Note: The concentration of HCl depends on the extent to which the resin is reduced. For example, 1% HCl was sufficient to strip Co-NTA MagBeads exposed to 1 mM DTT, 2% HCl for 5 mM DTT, and 3% for 10 mM DTT.
  3. Rinse the column with 10 bv dd water
  4. If the resin is not completely white, repeat steps 2 and 3. Otherwise, continue to step 4.
  5. Add 10 bv Wash Buffer and allow the majority of the volume to drip out of the column.
  6. Rinse the column with 10 bv dd water.
  7. Add 10 bv 10 mM CoSO4 to recharge the resin. Allow the volume to drip through the column by gravity.
  8. Rinse the column with 5 bv dd water
  9. Add 5 bv of Regeneration Buffer and incubate the matrix for 15 min at room temperature.
  10. Wash twice with 5 bv dd water each.
  11. Wash with 5 bv 20 mM Tris pH 8.0. Note: The extensive wash steps remove free nickel ions from the column, enhancing performance of the material in subsequent purifications, especially in presence of DTT.
  12. Wash twice with 5 bv dd water each
  13. . Add 10 bv 20% (v/v) ethanol and allow the majority of the volume to drip out of the column. The matrix is now ready to be re-used.
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1 ml 25% NTA magnetic beads, loaded with cobalt (II) chloride
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