Ni-NTA Agarose XL


Agarose Guide
Affinity chromatography is typically done with agarose beads with particle sizes of 40-150 µm. This material is very well suited for gravity flow and FPLC applications. Cube Biotech offers PureCube and PureCube 100 agaroses with a large variety of modification s that cover most applications. However, for specialized experiments, agaroses with exceptionally large bead sizes (300-500 µm in diameter) can be useful. For example, large beads settle fast by gravity and are therefore useful for automated purification procedures. Please note that due to the significantly different surface:volume ratio, binding capacity is usually lower than for standard sized beads. The polyhistidine tag is the most widely used affinity tag due to its small size, low immunogenicity, and versatility under native and denaturing conditions, as well as in presence of detergents and many other additives. Taking advantage of the affinity of transition metal ions for the imidazole ring of histidine, immobilized metal affinity chromatography (IMAC) is used to purify his-tagged proteins. Cube Biotech offers Ni-NTA Agarose XL. For loading with other transition metals, NTA Agarose XL is available on request.

PureCube Ni-NTA Agarose XL beads provide:

  • Automated protein purification: Large beads settle easily in 96 well plates
  • Purification of His-tagged proteins
  • binding capacity slightly lower than standard matrices ( <20 mg/mL)
  • Careful in-house production with high lot-to-lot reproducibility

Regenerated_Ni-NTA_wo_Legend_RGB72_01143
 
Fig. 1: PureCube Ni-NTA Agarose XL is robust against oxidation and regenerable. PureCube Ni-NTA Agarose was exposed to 5mM DTT for 1 h (A). After demonstrating that it could still bind GFP (B), the resin was washed, stripped (C), and reloaded with Ni2+ (D) following standard Cube protocol (see Cube Protocols & Datasheets).
 

Robust against oxidation and regenerable

PureCube Ni-NTA Agarose XL retains its color and function after exposure to as much as 10 mM DTT. Figure 3 shows a photo series of the resin after a 1 h exposure to 5 mM DTT. Unlike other resins, PureCube Ni-NTA Agarose did not turn brown (A). The resin was still able to bind GFP (B), with a measured binding capacity of 65 mg/mL (see Fig. 2). The resin could then be regenerated by stripping the NTA, turning the resin white (C), and reloading it with nickel ions (D). The protocol for regenerating PureCube Ni-NTA Agarose XL can be downloaded.

Features

Usage Specific binding and purification of 6x his-tagged proteins
Specifity Affinity to His-tagged proteins
Binding capacity >20 mg/mL
Chelator stability Stable in buffer containing 10 mM DTT and 1 mM EDTA
Filling quantity Delivered as a 50 % suspension
Bead size 300-500 μm
Bead Ligand Ni-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 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-NTA Agarose XL 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-NTA Agarose XL 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-NTA Agarose XL 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.
 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 Ni-NTA Agarose XL 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 Ni-NTA XL 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 NiSO4 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 Ni-NTA XL resin. 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 Ni-NTA XL 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 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 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.
  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.
Agarose Guide
Affinity chromatography is typically done with agarose beads with particle sizes of 40-150 µm. This material is very well suited for gravity flow and FPLC applications. Cube Biotech offers PureCube and PureCube 100 agaroses with a large variety of modification s that cover most applications. However, for specialized experiments, agaroses with exceptionally large bead sizes (300-500 µm in diameter) can be useful. For example, large beads settle fast by gravity and are therefore useful for automated purification procedures. Please note that due to the significantly different surface:volume ratio, binding capacity is usually lower than for standard sized beads. The polyhistidine tag is the most widely used affinity tag due to its small size, low immunogenicity, and versatility under native and denaturing conditions, as well as in presence of detergents and many other additives. Taking advantage of the affinity of transition metal ions for the imidazole ring of histidine, immobilized metal affinity chromatography (IMAC) is used to purify his-tagged proteins. Cube Biotech offers Ni-NTA Agarose XL. For loading with other transition metals, NTA Agarose XL is available on request.

PureCube Ni-NTA Agarose XL beads provide:

  • Automated protein purification: Large beads settle easily in 96 well plates
  • Purification of His-tagged proteins
  • binding capacity slightly lower than standard matrices ( <20 mg/mL)
  • Careful in-house production with high lot-to-lot reproducibility

Regenerated_Ni-NTA_wo_Legend_RGB72_01143
 
Fig. 1: PureCube Ni-NTA Agarose XL is robust against oxidation and regenerable. PureCube Ni-NTA Agarose was exposed to 5mM DTT for 1 h (A). After demonstrating that it could still bind GFP (B), the resin was washed, stripped (C), and reloaded with Ni2+ (D) following standard Cube protocol (see Cube Protocols & Datasheets).
 

Robust against oxidation and regenerable

PureCube Ni-NTA Agarose XL retains its color and function after exposure to as much as 10 mM DTT. Figure 3 shows a photo series of the resin after a 1 h exposure to 5 mM DTT. Unlike other resins, PureCube Ni-NTA Agarose did not turn brown (A). The resin was still able to bind GFP (B), with a measured binding capacity of 65 mg/mL (see Fig. 2). The resin could then be regenerated by stripping the NTA, turning the resin white (C), and reloading it with nickel ions (D). The protocol for regenerating PureCube Ni-NTA Agarose XL can be downloaded.

Features

Usage Specific binding and purification of 6x his-tagged proteins
Specifity Affinity to His-tagged proteins
Binding capacity >20 mg/mL
Chelator stability Stable in buffer containing 10 mM DTT and 1 mM EDTA
Filling quantity Delivered as a 50 % suspension
Bead size 300-500 μm
Bead Ligand Ni-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 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-NTA Agarose XL 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-NTA Agarose XL 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-NTA Agarose XL 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.
 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 Ni-NTA Agarose XL 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 Ni-NTA XL 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 NiSO4 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 Ni-NTA XL resin. 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 Ni-NTA XL 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 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 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.
  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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PureCube Ni-NTA Agarose XL PureCube Ni-NTA Agarose XL
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Ni-NTA Agarose XL
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PureCube Ni-NTA Agarose XL PureCube Ni-NTA Agarose XL
2 ml 50% Ni-NTA Agarose with medium particle size of 400 micrometer, for special applications
Article number: 55103
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