Purification of His-tagged proteins with Ni-NTA Agarose | Cube Biotech

Ni-NTA Agarose


MAGEBEADS
The His tag is the most widely used affinity tag due to its small size, low immunogenicity, and versatility under native or denaturing conditions, as well as in presence of detergents and many other additives. Cube Biotech offers high-performance PureCube Ni-NTA Agarose with 40 µm average bead size, based on BioWorks Workbeads, for purification of his-tagged proteins. PureCube Ni-NTA Agarose is provided as a 50% suspension, and also available as prepacked chromatography columns. For purification of his-tagged proteins from cell culture supernatants or for pull-down experiments, we recommend PureCube Ni-NTA MagBeads. To detect His-tagged proteins in Western Blot experiments, Cube Biotech offers the highly specific PentaHis antibody. Do you prefer Ni-IDA? We offer the same resin with that chelating ligand! (what's the difference?)
 

Why PureCube Ni-NTA Agarose?

Ni-NTA Agarose from Cube Biotech was successfully used in the following publications:

 ProteinYearAuthor
Plasmodium falciparum 6- phosphogluconate dehydrogenase 2018 Haeussler, K., Fritz-Wolf, K., Reichmann, M., Rahlfs, S., & Becker, K. 1
mCherry protein 2018 Wang, X., Liu, Y., Liu, J., & Chen, Z. 2
 Channelrhodopsin 2  2017  Volkov, O., Kovalev, K., Polovinkin, V., Borshchevskiy, V., Bamann, C., Astashkin, R., & Büldt, G. 3
 Geobacillus sp. Manganese catalase  2017  Li, H. C., Yu, Q., Wang, H., Cao, X. Y., Ma, L., & Li, Z. Q. 4
 Diacylglycerol kinase  2016  Rues, R. B., Henrich, E., Boland, C., Caffrey, M., & Bernhard, F. 5
 Glutamyl (aspartyl) specific aminopeptidase  2016  Stressler, T., Ewert, J., Merz, M., Funk, J., Claaßen, W., Lutz-Wahl, S., & Fischer, L. 6
 Histidine kinase domain of Ethylene Response Sensor 1  2016  Hsieh, Y. L., Lu, C. F., Chiang, B. Y., Liao, S. C., Chen, R. P. Y., Lin, C. S., ... & Yang, C. C. 7
 Arylsulfatase from Kluyveromyces lactis  2018  Stressler, T., Reichenberger, K., Glück, C., Leptihn, S., Pfannstiel, J., Swietalski, P., & Fischer, L. 8
 PepA from Lactobacillus delbrueckii  2017  Ewert, J., Glück, C., Strasdeit, H., Fischer, L., & Stressler, T. 9

Ni-NTAvsQ_vsG_BCapacity_wo_Legend_0114
 
Fig. 1: Over 20% more yield obtained with PureCube Ni-NTA Agarose. SDS-PAGE of GFP expressed in E. coli and purified in gravity columns with PureCube Ni-NTA Agarose and Ni-NTA resin from Competitor Q. 80 mg/mL protein yield was obtained with PureCube Ni-NTA Agarose (E1–E4, Cube) compared to 65 and 48 mg/mL, respectively, with the widely used alternative resins G and Q (E1–E4, Competitor G / Competitor Q).
 

High yield and purity

Our unique production process yields a Ni-NTA Agarose that exhibits a protein binding capacity >20% higher than that of two leading competitor products. Figure 1 shows the SDS-PAGE of GFP expressed in E. coli and purified in gravity colums with PureCube Ni-NTA Agarose and the Ni-NTA resin from Competitor G and Competitor Q. The protein yield in 4 elutions (E1-E4, Cube) was 80 mg/mL, compared to 65 and 48 mg/mL obtained with the alternative resins (E1-E4, Competitor G, Competitor Q). Similar results (10-18% higher binding capacity; data not shown here) were obtained comparing the purification of JNK1 (Kinase, 48 kDa) on PureCube Ni-NTA and the Ni-NTA of leading providers.

Ni-NTA_DTT&EDTAStability_Graph_wo_legend
 
Fig. 2: NTA is robust in the presence of reducing and chelating agents. GFP-His was purified on gravity columns containing PureCube Ni-NTA Agarose after exposing the resin for 1 h to 3 concentrations of DTT or EDTA. NTA exhibits a shallow decay rate in binding capacity.
 

Superior DTT and EDTA stability

PureCube Ni-NTA Agarose is very robust in the presence of DTT and EDTA. In a stability test, PureCube Ni-NTA Agarose was exposed to increasing concentrations of DTT or EDTA for 1 h. Thereafter, the resins were used to purify E. coli-expressed GFP-His in gravity columns. The binding capacity of the resin decreased in the presence of both DTT and EDTA but the decay rate was shallow. In presence of DTT, PureCube Ni-NTA Agarose lost on average 8% binding capacity with each increase in DTT concentration, resulting in an overall decay of 22% at 10 mM. Even at 1.5 mM EDTA, the resin still exihibits 54% of its maximum binding capacity (Fig. 2).

Regenerated_Ni-NTA_wo_Legend_RGB72_01143
 
Fig. 3: PureCube Ni-NTA Agarose 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 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 can be downloaded.

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-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

   
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 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 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 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.

Frequently asked questions

What are reasons for non specific binding?
Some histidine rich proteins can also bind to nickel. But washing with NaOH after elution of your protein of interest removes unspecific bound proteins from your resin.
 
I want to use high concentration of EDTA and DTT. Is it possible to use Ni-NTA from Cube Biotech?
No it is not recommended because nickel is reduced with DTT or dissolved with EDTA. If you want to use high concentrations of EDTA and DTT you should use our Indigo resin.
 
How is the capacity at high flow rates?
Due to the high ligand density of PURE Cube Ni-NTA agarose the material shows good performance even at high flow rates.
 
After using DTT my resin turned orange. How to regenerate it?
Read our detailed protocol for more information.
 
How much resin do I have to use?
That is depending on your expression level.

References

1. Haeussler, Kristina, et al. "Characterization of Plasmodium falciparum 6-phosphogluconate dehydrogenase as an antimalarial drug target." Journal of molecular biology 430.21 (2018): 4049-4067.
2. Wang, Xiaoliang, et al. "Protein–Polymer Microcapsules for PCR Technology." ChemBioChem 19.10 (2018): 1044-1048.
3. Volkov, Oleksandr, et al. "Structural insights into ion conduction by channelrhodopsin 2." Science 358.6366 (2017): eaan8862.
4. Li, Hai-Chao, et al. "A New Homo-Hexamer Mn-Containing Catalase from Geobacillus sp. WCH70." Catalysts 7.9 (2017): 277.
5. 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.
6. Stressler, Timo, et al. "A novel glutamyl (aspartyl)-specific aminopeptidase A from Lactobacillus delbrueckii with promising properties for application." PloS one 11.3 (2016): e0152139.
7. Hsieh, Yi-Lin, et al. "Molecular Characterization of Ethylene Response Sensor 1 (BoERS1) in Bambusa oldhamii." Plant molecular biology reporter 34.2 (2016): 387-398.
8. Stressler, Timo, et al. "A natural variant of arylsulfatase from Kluyveromyces lactis shows no formylglycine modification and has no enzyme activity." Applied microbiology and biotechnology102.6 (2018): 2709-2721.
9. Ewert, Jacob, et al. "Influence of the metal ion on the enzyme activity and kinetics of PepA from Lactobacillus delbrueckii." Enzyme and microbial technology 110 (2018): 69-78.
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Ni-NTA Agarose
MAGEBEADS
The His tag is the most widely used affinity tag due to its small size, low immunogenicity, and versatility under native or denaturing conditions, as well as in presence of detergents and many other additives. Cube Biotech offers high-performance PureCube Ni-NTA Agarose with 40 µm average bead size, based on BioWorks Workbeads, for purification of his-tagged proteins. PureCube Ni-NTA Agarose is provided as a 50% suspension, and also available as prepacked chromatography columns. For purification of his-tagged proteins from cell culture supernatants or for pull-down experiments, we recommend PureCube Ni-NTA MagBeads. To detect His-tagged proteins in Western Blot experiments, Cube Biotech offers the highly specific PentaHis antibody. Do you prefer Ni-IDA? We offer the same resin with that chelating ligand! (what's the difference?)
 

Why PureCube Ni-NTA Agarose?

Ni-NTA Agarose from Cube Biotech was successfully used in the following publications:

 ProteinYearAuthor
Plasmodium falciparum 6- phosphogluconate dehydrogenase 2018 Haeussler, K., Fritz-Wolf, K., Reichmann, M., Rahlfs, S., & Becker, K. 1
mCherry protein 2018 Wang, X., Liu, Y., Liu, J., & Chen, Z. 2
 Channelrhodopsin 2  2017  Volkov, O., Kovalev, K., Polovinkin, V., Borshchevskiy, V., Bamann, C., Astashkin, R., & Büldt, G. 3
 Geobacillus sp. Manganese catalase  2017  Li, H. C., Yu, Q., Wang, H., Cao, X. Y., Ma, L., & Li, Z. Q. 4
 Diacylglycerol kinase  2016  Rues, R. B., Henrich, E., Boland, C., Caffrey, M., & Bernhard, F. 5
 Glutamyl (aspartyl) specific aminopeptidase  2016  Stressler, T., Ewert, J., Merz, M., Funk, J., Claaßen, W., Lutz-Wahl, S., & Fischer, L. 6
 Histidine kinase domain of Ethylene Response Sensor 1  2016  Hsieh, Y. L., Lu, C. F., Chiang, B. Y., Liao, S. C., Chen, R. P. Y., Lin, C. S., ... & Yang, C. C. 7
 Arylsulfatase from Kluyveromyces lactis  2018  Stressler, T., Reichenberger, K., Glück, C., Leptihn, S., Pfannstiel, J., Swietalski, P., & Fischer, L. 8
 PepA from Lactobacillus delbrueckii  2017  Ewert, J., Glück, C., Strasdeit, H., Fischer, L., & Stressler, T. 9

Ni-NTAvsQ_vsG_BCapacity_wo_Legend_0114
 
Fig. 1: Over 20% more yield obtained with PureCube Ni-NTA Agarose. SDS-PAGE of GFP expressed in E. coli and purified in gravity columns with PureCube Ni-NTA Agarose and Ni-NTA resin from Competitor Q. 80 mg/mL protein yield was obtained with PureCube Ni-NTA Agarose (E1–E4, Cube) compared to 65 and 48 mg/mL, respectively, with the widely used alternative resins G and Q (E1–E4, Competitor G / Competitor Q).
 

High yield and purity

Our unique production process yields a Ni-NTA Agarose that exhibits a protein binding capacity >20% higher than that of two leading competitor products. Figure 1 shows the SDS-PAGE of GFP expressed in E. coli and purified in gravity colums with PureCube Ni-NTA Agarose and the Ni-NTA resin from Competitor G and Competitor Q. The protein yield in 4 elutions (E1-E4, Cube) was 80 mg/mL, compared to 65 and 48 mg/mL obtained with the alternative resins (E1-E4, Competitor G, Competitor Q). Similar results (10-18% higher binding capacity; data not shown here) were obtained comparing the purification of JNK1 (Kinase, 48 kDa) on PureCube Ni-NTA and the Ni-NTA of leading providers.

Ni-NTA_DTT&EDTAStability_Graph_wo_legend
 
Fig. 2: NTA is robust in the presence of reducing and chelating agents. GFP-His was purified on gravity columns containing PureCube Ni-NTA Agarose after exposing the resin for 1 h to 3 concentrations of DTT or EDTA. NTA exhibits a shallow decay rate in binding capacity.
 

Superior DTT and EDTA stability

PureCube Ni-NTA Agarose is very robust in the presence of DTT and EDTA. In a stability test, PureCube Ni-NTA Agarose was exposed to increasing concentrations of DTT or EDTA for 1 h. Thereafter, the resins were used to purify E. coli-expressed GFP-His in gravity columns. The binding capacity of the resin decreased in the presence of both DTT and EDTA but the decay rate was shallow. In presence of DTT, PureCube Ni-NTA Agarose lost on average 8% binding capacity with each increase in DTT concentration, resulting in an overall decay of 22% at 10 mM. Even at 1.5 mM EDTA, the resin still exihibits 54% of its maximum binding capacity (Fig. 2).

Regenerated_Ni-NTA_wo_Legend_RGB72_01143
 
Fig. 3: PureCube Ni-NTA Agarose 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 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 can be downloaded.

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-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

   
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 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 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 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.

Frequently asked questions

What are reasons for non specific binding?
Some histidine rich proteins can also bind to nickel. But washing with NaOH after elution of your protein of interest removes unspecific bound proteins from your resin.
 
I want to use high concentration of EDTA and DTT. Is it possible to use Ni-NTA from Cube Biotech?
No it is not recommended because nickel is reduced with DTT or dissolved with EDTA. If you want to use high concentrations of EDTA and DTT you should use our Indigo resin.
 
How is the capacity at high flow rates?
Due to the high ligand density of PURE Cube Ni-NTA agarose the material shows good performance even at high flow rates.
 
After using DTT my resin turned orange. How to regenerate it?
Read our detailed protocol for more information.
 
How much resin do I have to use?
That is depending on your expression level.

References

1. Haeussler, Kristina, et al. "Characterization of Plasmodium falciparum 6-phosphogluconate dehydrogenase as an antimalarial drug target." Journal of molecular biology 430.21 (2018): 4049-4067.
2. Wang, Xiaoliang, et al. "Protein–Polymer Microcapsules for PCR Technology." ChemBioChem 19.10 (2018): 1044-1048.
3. Volkov, Oleksandr, et al. "Structural insights into ion conduction by channelrhodopsin 2." Science 358.6366 (2017): eaan8862.
4. Li, Hai-Chao, et al. "A New Homo-Hexamer Mn-Containing Catalase from Geobacillus sp. WCH70." Catalysts 7.9 (2017): 277.
5. 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.
6. Stressler, Timo, et al. "A novel glutamyl (aspartyl)-specific aminopeptidase A from Lactobacillus delbrueckii with promising properties for application." PloS one 11.3 (2016): e0152139.
7. Hsieh, Yi-Lin, et al. "Molecular Characterization of Ethylene Response Sensor 1 (BoERS1) in Bambusa oldhamii." Plant molecular biology reporter 34.2 (2016): 387-398.
8. Stressler, Timo, et al. "A natural variant of arylsulfatase from Kluyveromyces lactis shows no formylglycine modification and has no enzyme activity." Applied microbiology and biotechnology102.6 (2018): 2709-2721.
9. Ewert, Jacob, et al. "Influence of the metal ion on the enzyme activity and kinetics of PepA from Lactobacillus delbrueckii." Enzyme and microbial technology 110 (2018): 69-78.
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