Ni-IDA Agarose


Purification resins
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. Cube Biotech offers high-performance PureCube Ni-IDA Agarose, based on BioWorks Workbeads, for purification of his-tagged proteins. To detect His-tagged proteins in Western Blot experiments, Cube Biotech offers tag-specific antibodies. 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.

Why PureCube Ni-IDA Agarose?

  • Binding capacity of up to 50 mg/mL
  • Stable under commonly used concentrations of DTT and EDTA
  • Can be regenerated for reuse

Our Ni-IDA Resins in detail

Ni_binding_capaticity
Fig. 1: The binding capacity of PureCube Ni-IDA Agarose is comparable to resins from a leading supplier. SDS-PAGE of CAT purified in gravity columns with PureCube Ni-IDA Agarose and Ni resin from Competitor G show elution fractions (E1-E5) with similar protein yields.
Strong and robust performance
Based on the same agarose matrix with high porosity and physical stability, PureCube Ni-IDA agarose exhibits a protein capacity of 50 mg protein per mL resin, which is competitive with that of products from market-leading providers. Figure 1 shows the SDS-PAGE analysis of 6-His chloramphenicoltransferase (CAT) expressed in E. coli and purified on gravity flow columns filled with PureCUbe Ni-IDA Agarose or Competitor G Ni-Sepharose High Performance resin. The amount of protein drawn down from the columns in 5 elution fractions was comparable for the two affinity resins (CL: cleared lysate; FT: flow-through; W1-2: wash fractions; E1-5: elution fractions). Table 1 compares additional parameters among PureCube Ni-IDA Agarose and two equivalent market-leading resins.
Table 1. Technical and performance parameters of 3 Ni-IDA agarose resins.
 Cube BiotechCompetitor GCompetitor S
Particle size 32-60µm average 90 µm 45-160 µm
Metal ion capacity (Cu, Ni) >25 µmol/mL >30 µmol/mL 6-18 µmol/mL
Binding capacity 50 mg/mL > 40 mg/ml 15 mg/mL
pH stability (processing) 3.0-12.0 3.0-13.0 No information
Recommended flow rate 0.5-2.0 mL/min (6.0 mL/min poss.) 1.0 mL/min 1.0 mL/min
DTT stability 10 mM No information 5 mM
EDTA stability 1.0 mM No information Not recommended
Top-quality materials, competitive performance
PureCube Ni-IDA Agarose is produced with a highly cross-linked agarose which is physically very stable and suitable for purification processes under low pressure (bulk purifications as well as packed in chromatography columns). Additionally, the high porosity and uniform size of the matrix allows for optimal protein interaction and high reproducibility between individual purification runs. Table 1 summarizes technical and performance parameters of PureCube Ni-IDA Agarose and 2 equivalent, market-leading resins.

Features

Usage Specific binding and purification of 6x his-tagged proteins
Specifity Affinity to His-tagged proteins
Binding capacity >70 mg/mL
Chelator stability Stable in buffer containing 10 mM DTT and 1 mM EDTA
Filling quantity Delivered as a 50 % suspension
Bead size 40 μm
Bead Ligand Ni-IDA
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 and buffer compositions are also avaible as PDF-Files on the Protocols & Datasheets page.
   
A.Protocol for purification under native conditions:
 
 
  1. Thaw the E. coli cell pellets corresponding to 200 mL bacterial culture on ice for 15 min. 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 10 mL Native Lysis Buffer supplemented with 1 mg/mL lysozyme, and pour it into a 50 mL conical centrifuge tube.
  3. If the solution is very viscous, add 3 units Benzonase® per mL E.coli culture volume to the lysis buffer. Alternatively or additionally, sonicate the lysate to improve cell disruption.
  4. Incubate on an end-over-end shaker at room temperature for 30 min, or at 4 °C for 1 h, depending on the temperature stability of the protein.
  5. Centrifuge the lysate for 30 min at 10,000 x g and 2-8 °C. Carefully collect the supernatant without touching the pellet. Note: The supernatant contains the cleared lysate fraction. We recommend to take aliquots of all fractions for SDS-PAGE analysis.
  6. Resuspend the PureCube Ni-IDA Agarose by inverting the bottle until the suspension is homogeneous. Transfer 1 mL of the 50 % suspension (corresponding to 500 μL bed volume) to a 15 mL conical centrifuge tube. Allow the resin to settle by gravity and remove the supernatant. Tip: Alternatively, resin equilibration can be performed directly in the disposable gravity flow column.
  7. Add 2.5 mL Native Lysis Buffer and gently resuspend the slurry to equilibrate the resin. Allow the resin to settle by gravity and remove 2 mL supernatant.
  8. Add 10 mL cleared lysate to the equilibrated PureCube Ni-IDA Agarose resin and incubate at 4 °C for 1 h on an end-over-end shaker. Tip: Alternatively, batch binding can be performed directly in a gravity flow column with closed bottom and top outlets.
  9. Transfer the binding suspension to a disposable gravity flow column with a capped bottom outlet. Use Lysis Buffer to rinse the centrifuge tube and remove resin adhered to the wall.
  10. Remove the bottom cap of the column and collect the flow-through.
  11. Wash the column with 5 mL Native Wash Buffer. Repeat the washing step at least 3 times.
  12. Elute the His-tagged protein 5 times using 0.5 mL Native Elution Buffer. Collect each eluate in a separate tube and determine the protein concentration of each fraction. Optional: Incubate the resin for 15 min in Elution Buffer before collecting the eluate to increase protein yields.
  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.
 B.Protocol for purification under denaturing conditions:
 
 
  1. Thaw the E. coli cell pellet on ice.
  2. Resuspend the cell pellet in 10 mL Denaturing Lysis Buffer. Optional: Benzonase® can be added to the lysate to reduce viscosity caused by nucleic acids (3 U/mL bacterial culture). Nucleic acids can also 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. Resuspend the PureCube Ni-IDA Agarose by inverting the bottle until the suspension is homogeneous. Transfer 1 mL of the 50% suspension (corresponding to 0.5 mL bed volume) into a 15 mL conical centrifuge tube. Allow the resin to settle by gravity and remove the supernatant.
  6. Add the cleared lysate to the resin and incubate the mixture for 1 h at room temperature on an end-over-end shaker. Tip: Alternatively, batch binding can be done directly in a gravity flow column with closed top and bottom outlet.
  7. Transfer the binding suspension to a disposable gravity flow column with a capped bottom outlet. Use Lysis Buffer to rinse the centrifuge tube and remove resin adhered to the wall.
  8. Remove the bottom cap of the column and collect the flow-through.
  9. Wash the column with 5 mL Denaturing Wash Buffer. Repeat the washing step at least 3 times.
  10. Elute the His-tagged protein 5 times using 0.5 mL Denaturing Elution Buffer. Collect each eluate in a separate tube and determine the protein concentration of each fraction. Tip: If the target protein is acid-labile, elution can be performed with 250-500 mM imidazole.
  11. 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.
  12. Optional: Perform Western Blot experiment using PentaHis Antibody.
Purification resins
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. Cube Biotech offers high-performance PureCube Ni-IDA Agarose, based on BioWorks Workbeads, for purification of his-tagged proteins. To detect His-tagged proteins in Western Blot experiments, Cube Biotech offers tag-specific antibodies. 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.

Why PureCube Ni-IDA Agarose?

  • Binding capacity of up to 50 mg/mL
  • Stable under commonly used concentrations of DTT and EDTA
  • Can be regenerated for reuse

Our Ni-IDA Resins in detail

Ni_binding_capaticity
Fig. 1: The binding capacity of PureCube Ni-IDA Agarose is comparable to resins from a leading supplier. SDS-PAGE of CAT purified in gravity columns with PureCube Ni-IDA Agarose and Ni resin from Competitor G show elution fractions (E1-E5) with similar protein yields.
Strong and robust performance
Based on the same agarose matrix with high porosity and physical stability, PureCube Ni-IDA agarose exhibits a protein capacity of 50 mg protein per mL resin, which is competitive with that of products from market-leading providers. Figure 1 shows the SDS-PAGE analysis of 6-His chloramphenicoltransferase (CAT) expressed in E. coli and purified on gravity flow columns filled with PureCUbe Ni-IDA Agarose or Competitor G Ni-Sepharose High Performance resin. The amount of protein drawn down from the columns in 5 elution fractions was comparable for the two affinity resins (CL: cleared lysate; FT: flow-through; W1-2: wash fractions; E1-5: elution fractions). Table 1 compares additional parameters among PureCube Ni-IDA Agarose and two equivalent market-leading resins.
Table 1. Technical and performance parameters of 3 Ni-IDA agarose resins.
 Cube BiotechCompetitor GCompetitor S
Particle size 32-60µm average 90 µm 45-160 µm
Metal ion capacity (Cu, Ni) >25 µmol/mL >30 µmol/mL 6-18 µmol/mL
Binding capacity 50 mg/mL > 40 mg/ml 15 mg/mL
pH stability (processing) 3.0-12.0 3.0-13.0 No information
Recommended flow rate 0.5-2.0 mL/min (6.0 mL/min poss.) 1.0 mL/min 1.0 mL/min
DTT stability 10 mM No information 5 mM
EDTA stability 1.0 mM No information Not recommended
Top-quality materials, competitive performance
PureCube Ni-IDA Agarose is produced with a highly cross-linked agarose which is physically very stable and suitable for purification processes under low pressure (bulk purifications as well as packed in chromatography columns). Additionally, the high porosity and uniform size of the matrix allows for optimal protein interaction and high reproducibility between individual purification runs. Table 1 summarizes technical and performance parameters of PureCube Ni-IDA Agarose and 2 equivalent, market-leading resins.

Features

Usage Specific binding and purification of 6x his-tagged proteins
Specifity Affinity to His-tagged proteins
Binding capacity >70 mg/mL
Chelator stability Stable in buffer containing 10 mM DTT and 1 mM EDTA
Filling quantity Delivered as a 50 % suspension
Bead size 40 μm
Bead Ligand Ni-IDA
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 and buffer compositions are also avaible as PDF-Files on the Protocols & Datasheets page.
   
A.Protocol for purification under native conditions:
 
 
  1. Thaw the E. coli cell pellets corresponding to 200 mL bacterial culture on ice for 15 min. 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 10 mL Native Lysis Buffer supplemented with 1 mg/mL lysozyme, and pour it into a 50 mL conical centrifuge tube.
  3. If the solution is very viscous, add 3 units Benzonase® per mL E.coli culture volume to the lysis buffer. Alternatively or additionally, sonicate the lysate to improve cell disruption.
  4. Incubate on an end-over-end shaker at room temperature for 30 min, or at 4 °C for 1 h, depending on the temperature stability of the protein.
  5. Centrifuge the lysate for 30 min at 10,000 x g and 2-8 °C. Carefully collect the supernatant without touching the pellet. Note: The supernatant contains the cleared lysate fraction. We recommend to take aliquots of all fractions for SDS-PAGE analysis.
  6. Resuspend the PureCube Ni-IDA Agarose by inverting the bottle until the suspension is homogeneous. Transfer 1 mL of the 50 % suspension (corresponding to 500 μL bed volume) to a 15 mL conical centrifuge tube. Allow the resin to settle by gravity and remove the supernatant. Tip: Alternatively, resin equilibration can be performed directly in the disposable gravity flow column.
  7. Add 2.5 mL Native Lysis Buffer and gently resuspend the slurry to equilibrate the resin. Allow the resin to settle by gravity and remove 2 mL supernatant.
  8. Add 10 mL cleared lysate to the equilibrated PureCube Ni-IDA Agarose resin and incubate at 4 °C for 1 h on an end-over-end shaker. Tip: Alternatively, batch binding can be performed directly in a gravity flow column with closed bottom and top outlets.
  9. Transfer the binding suspension to a disposable gravity flow column with a capped bottom outlet. Use Lysis Buffer to rinse the centrifuge tube and remove resin adhered to the wall.
  10. Remove the bottom cap of the column and collect the flow-through.
  11. Wash the column with 5 mL Native Wash Buffer. Repeat the washing step at least 3 times.
  12. Elute the His-tagged protein 5 times using 0.5 mL Native Elution Buffer. Collect each eluate in a separate tube and determine the protein concentration of each fraction. Optional: Incubate the resin for 15 min in Elution Buffer before collecting the eluate to increase protein yields.
  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.
 B.Protocol for purification under denaturing conditions:
 
 
  1. Thaw the E. coli cell pellet on ice.
  2. Resuspend the cell pellet in 10 mL Denaturing Lysis Buffer. Optional: Benzonase® can be added to the lysate to reduce viscosity caused by nucleic acids (3 U/mL bacterial culture). Nucleic acids can also 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. Resuspend the PureCube Ni-IDA Agarose by inverting the bottle until the suspension is homogeneous. Transfer 1 mL of the 50% suspension (corresponding to 0.5 mL bed volume) into a 15 mL conical centrifuge tube. Allow the resin to settle by gravity and remove the supernatant.
  6. Add the cleared lysate to the resin and incubate the mixture for 1 h at room temperature on an end-over-end shaker. Tip: Alternatively, batch binding can be done directly in a gravity flow column with closed top and bottom outlet.
  7. Transfer the binding suspension to a disposable gravity flow column with a capped bottom outlet. Use Lysis Buffer to rinse the centrifuge tube and remove resin adhered to the wall.
  8. Remove the bottom cap of the column and collect the flow-through.
  9. Wash the column with 5 mL Denaturing Wash Buffer. Repeat the washing step at least 3 times.
  10. Elute the His-tagged protein 5 times using 0.5 mL Denaturing Elution Buffer. Collect each eluate in a separate tube and determine the protein concentration of each fraction. Tip: If the target protein is acid-labile, elution can be performed with 250-500 mM imidazole.
  11. 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.
  12. Optional: Perform Western Blot experiment using PentaHis Antibody.
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PureCube Ni-IDA Agarose PureCube Ni-IDA Agarose
Article number: 30103
Ni-IDA Agarose
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PureCube Ni-IDA Agarose PureCube Ni-IDA Agarose
2 ml 50% IDA Agarose, loaded with nickel (II) sulfate
Article number: 30103
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