Ni-IDA 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. PureCube Ni-IDA MagBeads are ferrimagnetic agarose beads coupled to an IDA chelating ligand, loaded with nickel ions. PureCube Ni-NTA MagBeads are delivered as a 25% suspension. For increased specificity, we offer a range of different His affinity resins, e.g. carrying the NTA ligand (what's the difference?). Please see our overview page for more information.

PureCube Ni-IDA MagBeads offer:

  • High binding capacity (up to 70 mg/mL settled beads)
  • Low unspecific binding
  • Stable in up to 10 mM DTT and up to 1 mM EDTA (Not stable enough?)
  • Ideal for purification from dilute solutions and for pulldown experiments
  • Also available as Ni-IDA Agarose and prepacked cartridges

Ni-IDA Magnetic Beads from Cube Biotech were successfully used in the following publications:

 ProteinYearAuthor
UGDH, NOX, GalK, USP, GalT and GlcAT 2019 Heinzler R., Fischöder T., Elling L., Franzreb M.1

Features

Usage Specific binding and purification of his-tagged proteins
Specifity Affinity to His-tagged proteins
High binding capacity 40 mg/mL settled beads
Compability pH 2-14, 100% methanol, 100% ethanol, 8 M urea, 6 M guanidinium hydrochloride, 30% (v/v) acetonitrile
Filling quantity Delivered as a 25 % suspension
Bead size 30 μm
Bead Ligand Ni-IDA
Required equipment
 

Applications

All protocols and buffer compositions are also avaible as PDF-Files on the Protocols & Datasheets page.
   
A.Protein purification protocol for 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. IDA MagBead tolerance of EDTA is 1 mM and 10 mM for DTT.
  3. Incubate at room temperature for 30 min on an end-overend 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 His Affinity MagBeads by vortexing. Transfer 40 μL of the 25% magnetic beads suspension onto the lysate (or the volume adjusted to expression level; see Table 1).
  7. Incubate the lysate-magnetic bead mixture at room temperature for 1 h on an end-over-end shaker. Tip: Briefly centrifuge the sample before placing it on the magnetic separator in order to collect liquid from the lid.
  8. Place the tube on the magnetic microtube stand until the beads separate and remove the supernatant. This is the flow-through fraction.
  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. These are the wash fractions.
  10. Repeat step 9 twice.
  11. Elute the His-tagged protein using 100 μL Denaturing Elution Buffer (or the volume adjusted to the expression level. Tip: If the target protein is acid-labile, elution can be perfored with 250-500 mM imidazole.
  12. . Repeat step 11. Collect each elution fraction in a separate tube and determine the protein concentration of each fraction. These are the elution fractions
  13. 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.
  14. Optional: Perform Western Blot experiment using PentaHis Antibody. Note: Do not boil membrane proteins. Instead, incubate samples at 46˚C for 30 min in preparation for SDS-PAGE analysis.
   
B.Protein purification protocol for 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 Lysis Buffer supplemented with 1 mg/mL lysozyme. Optional: Add 1 tablet protease inhibitor cocktail to the Lysis Buffer. IDA MagBead tolerance of EDTA is 1 mM and 10 mM for DTT.
  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. Tip: Lysis Buffer contains 10 mM imidazole to prevent binding of untagged proteins. If His-tagged proteins do not bind under these conditions, reduce the imidazole concentration to 1–5 mM.
  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 His Affinity MagBeads by vortexing. Transfer 40 μL of the 25% magnetic bead suspension into a conical microcentrifuge tube (or the volume adjusted to the expression level).
  7. Add 500 µL 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. This is the flow-through fraction
  10. Remove the tube from the magnet. Add 500 µL Wash Buffer and mix by vortexing. Place the tube again on the magnetic microtube stand and allow the beads to separate. Remove the supernatant. These are the wash fractions.
  11. Repeat step 10 twice.
  12. Elute the His-tagged protein using 100 μL Elution Buffer (or the volume adjusted to the expression level). These are the elution fractions.
  13. Repeat step 12. Collect each elution fraction in a separate tube and determine the protein concentration of each fraction. Note: Do not boil membrane proteins. Instead, incubate the sample at 46˚C for 30 min in preparation for SDS-PAGE analysis.
  14. Analyze all fractions by SDS-PAGE.
  15. Optional: Perform Western Blot experiment using PentaHis Antibody.
C.Wash Protocol (recommended after each run)
 
 
  1. Remove the majority of the fluid in the column containing the Ni-NTA or Ni-IDA 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 (recommended after each run)
 
 
  1. Remove the majority of the fluid in the column containing the Ni-NTA or Ni-IDA matrix. Add 10 bv dd water and allow the majority of the volume to drip out of the column.
  2. Add 10 bv 100 mM EDTA to the column and allow the entire volume to flow through the matrix. 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. Rinse the column again with 10 bv dd water.
  4. Add 10 bv Wash Buffer to the column and allow the entire volume to flow through the matrix.
  5. Rinse the column with 10 bv dd water
  6. Add 10 bv 10m M NiSO4 to recharge the matrix. Allow the volume to drip through the column by gravity.
  7. Rinse the column with 5 bv dd water.
  8. Add 5 bv of Regeneration Buffer and incubate the column for 15 min at room temperature.
  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 reducing agents like DTT (recommended after each run)
 
 
  1. Remove the majority of the fluid in the column containing the Ni-NTA or Ni-IDA resin. Add 10 bv dd water and allow the majority of the volume to drip out of the column. Note: Resins exposed to reducing agents should always be regenerated after a run.
  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 Ni-NTA and Ni-IDA resin 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 5.
  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 NiSO4 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. 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
  11. Wash with 5 bv 20 mM Tris pH 8.0.
  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.

References

1. Heinzler, R. et al. (2019). Toward Automated Enzymatic Glycan Synthesis in a Compartmented Flow Microreactor System. Advanced Synthesis & Catalysis. 10.1002/adsc.201900709.
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. PureCube Ni-IDA MagBeads are ferrimagnetic agarose beads coupled to an IDA chelating ligand, loaded with nickel ions. PureCube Ni-NTA MagBeads are delivered as a 25% suspension. For increased specificity, we offer a range of different His affinity resins, e.g. carrying the NTA ligand (what's the difference?). Please see our overview page for more information.

PureCube Ni-IDA MagBeads offer:

  • High binding capacity (up to 70 mg/mL settled beads)
  • Low unspecific binding
  • Stable in up to 10 mM DTT and up to 1 mM EDTA (Not stable enough?)
  • Ideal for purification from dilute solutions and for pulldown experiments
  • Also available as Ni-IDA Agarose and prepacked cartridges

Ni-IDA Magnetic Beads from Cube Biotech were successfully used in the following publications:

 ProteinYearAuthor
UGDH, NOX, GalK, USP, GalT and GlcAT 2019 Heinzler R., Fischöder T., Elling L., Franzreb M.1

Features

Usage Specific binding and purification of his-tagged proteins
Specifity Affinity to His-tagged proteins
High binding capacity 40 mg/mL settled beads
Compability pH 2-14, 100% methanol, 100% ethanol, 8 M urea, 6 M guanidinium hydrochloride, 30% (v/v) acetonitrile
Filling quantity Delivered as a 25 % suspension
Bead size 30 μm
Bead Ligand Ni-IDA
Required equipment
 

Applications

All protocols and buffer compositions are also avaible as PDF-Files on the Protocols & Datasheets page.
   
A.Protein purification protocol for 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. IDA MagBead tolerance of EDTA is 1 mM and 10 mM for DTT.
  3. Incubate at room temperature for 30 min on an end-overend 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 His Affinity MagBeads by vortexing. Transfer 40 μL of the 25% magnetic beads suspension onto the lysate (or the volume adjusted to expression level; see Table 1).
  7. Incubate the lysate-magnetic bead mixture at room temperature for 1 h on an end-over-end shaker. Tip: Briefly centrifuge the sample before placing it on the magnetic separator in order to collect liquid from the lid.
  8. Place the tube on the magnetic microtube stand until the beads separate and remove the supernatant. This is the flow-through fraction.
  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. These are the wash fractions.
  10. Repeat step 9 twice.
  11. Elute the His-tagged protein using 100 μL Denaturing Elution Buffer (or the volume adjusted to the expression level. Tip: If the target protein is acid-labile, elution can be perfored with 250-500 mM imidazole.
  12. . Repeat step 11. Collect each elution fraction in a separate tube and determine the protein concentration of each fraction. These are the elution fractions
  13. 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.
  14. Optional: Perform Western Blot experiment using PentaHis Antibody. Note: Do not boil membrane proteins. Instead, incubate samples at 46˚C for 30 min in preparation for SDS-PAGE analysis.
   
B.Protein purification protocol for 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 Lysis Buffer supplemented with 1 mg/mL lysozyme. Optional: Add 1 tablet protease inhibitor cocktail to the Lysis Buffer. IDA MagBead tolerance of EDTA is 1 mM and 10 mM for DTT.
  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. Tip: Lysis Buffer contains 10 mM imidazole to prevent binding of untagged proteins. If His-tagged proteins do not bind under these conditions, reduce the imidazole concentration to 1–5 mM.
  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 His Affinity MagBeads by vortexing. Transfer 40 μL of the 25% magnetic bead suspension into a conical microcentrifuge tube (or the volume adjusted to the expression level).
  7. Add 500 µL 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. This is the flow-through fraction
  10. Remove the tube from the magnet. Add 500 µL Wash Buffer and mix by vortexing. Place the tube again on the magnetic microtube stand and allow the beads to separate. Remove the supernatant. These are the wash fractions.
  11. Repeat step 10 twice.
  12. Elute the His-tagged protein using 100 μL Elution Buffer (or the volume adjusted to the expression level). These are the elution fractions.
  13. Repeat step 12. Collect each elution fraction in a separate tube and determine the protein concentration of each fraction. Note: Do not boil membrane proteins. Instead, incubate the sample at 46˚C for 30 min in preparation for SDS-PAGE analysis.
  14. Analyze all fractions by SDS-PAGE.
  15. Optional: Perform Western Blot experiment using PentaHis Antibody.
C.Wash Protocol (recommended after each run)
 
 
  1. Remove the majority of the fluid in the column containing the Ni-NTA or Ni-IDA 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 (recommended after each run)
 
 
  1. Remove the majority of the fluid in the column containing the Ni-NTA or Ni-IDA matrix. Add 10 bv dd water and allow the majority of the volume to drip out of the column.
  2. Add 10 bv 100 mM EDTA to the column and allow the entire volume to flow through the matrix. 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. Rinse the column again with 10 bv dd water.
  4. Add 10 bv Wash Buffer to the column and allow the entire volume to flow through the matrix.
  5. Rinse the column with 10 bv dd water
  6. Add 10 bv 10m M NiSO4 to recharge the matrix. Allow the volume to drip through the column by gravity.
  7. Rinse the column with 5 bv dd water.
  8. Add 5 bv of Regeneration Buffer and incubate the column for 15 min at room temperature.
  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 reducing agents like DTT (recommended after each run)
 
 
  1. Remove the majority of the fluid in the column containing the Ni-NTA or Ni-IDA resin. Add 10 bv dd water and allow the majority of the volume to drip out of the column. Note: Resins exposed to reducing agents should always be regenerated after a run.
  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 Ni-NTA and Ni-IDA resin 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 5.
  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 NiSO4 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. 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
  11. Wash with 5 bv 20 mM Tris pH 8.0.
  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.

References

1. Heinzler, R. et al. (2019). Toward Automated Enzymatic Glycan Synthesis in a Compartmented Flow Microreactor System. Advanced Synthesis & Catalysis. 10.1002/adsc.201900709.
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PureCube Ni-IDA MagBeads PureCube Ni-IDA MagBeads
Article number: 30201
Ni-IDA MagBeads
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PureCube Ni-IDA MagBeads PureCube Ni-IDA MagBeads
1 ml 25% IDA magnetic beads, loaded with nickel (II) sulfate
Article number: 30201
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