100 INDIGO Ni-Agarose


protein_purification_resins
The His tag is the most widely used affinity tag due to its small size, low immuno genicity, and versatility under native or denaturing conditions, as well as in presence of detergents and many other additives. Cube Biotech introduces the novel PureCube 100 INDIGO Ni-Agarose, based on high quality crosslinked agarose with 100 µm mean diameter, providing high flow rates in batch and FPLC applications. Using the novel, proprietary INDIGO ligand, purification of his-tagged proteins is possible in the presence of up to 20 mM DTT and 20 mM EDTA (Fig.2) at a protein capacity considerably higher than for comparable competitor products (Fig.3). The novel INDIGO ligand cannot be stripped with EDTA. However, without any regeneration, PureCube 100 INDIGO Ni-Agarose was re-used eight times without reduction in performance (Fig.4). PureCube 100 INDIGO Ni-Agarose is provided as a 50% suspension. To detect His-tagged proteins in Western Blot experiments, Cube Biotech offers the highly specific PentaHis antibody.

Why PureCube 100 INDIGO Ni-Agarose?

  • Stable in 20 mM DTT and 20 mM EDTA, at pH 2-13
  • High yield (up to 80 mg/mL) and purity
  • Compatible with eukaryotic cell culture media
  • Also available as INDIGO Ni-MagBeads

Purity and affinity - superior to our competitors

The purification of proteins always involves the balance act between purity and affinity of your protein. This is especially important when working with the HIS tag. The binding of his tagged proteins to the metal ion of the agarose resin or magnetic beads is based on electric charges. and not distinct peptide sequences. Therefore it tends to impurity as other moleculed in the cell can also be slightly charged similar to a poly his tag.
Our R&D Team however managed to overcome this issue: We developed a new ligand that we named INDIGO, due to its color. Besides its highly superior EDTA and DTT stability (more to that later), its affinity is on par with traditional Ni-Agarose resins, while simultaneously having a highly increased specificity and therefore purity (see figure 1).
High specificity with low expressed proteins
This is especially important when working with low expressed proteins. Low protein amounts lead to highly impure protein purifications with traditional Ni-Agarose as many agarose beads remain uncovered by the protein of interest due to its low abundance. Unspecific binding of unwanted peptides then occurs at these beads that subsequent lead to impurities later on (see figure 1). INDIGO-Ni agarose overcomes this issue, because even at low proteins of interest concentrations in the cell the binding of the his tag to the agarose beads remains highly specific (figure 1).
INDIGO Purification qualities
Fig. 1: Overview of INDIGO-Ni agarose resin's purification properties. The immensly superior puritiy compared to traditional Ni-NTA agarose is worth mentioning. Especially for low expressing proteins. Left side: The performance of our PureCube 100 INDIGO resin. Right side: Performance of Ni-NTA agarose from competitor T.
For this demonstration his tagged GFP was added in known concentrations (see bottom of the blot) to an E.coli cell lysate. This was done to mimic a low protein expression rates with different distinct protein concentrations. As it can be seen both Ni-NTA from competitor T and INDIGO-Ni agarose resin purify his tagged GFP, even at very low concentrations. However the INDIGO concentration is highly superior in purity.
To put it simple: If your protein of interest (POI) is as rare as the famous needle in the haystack, traditional NI-agarose gives you the POI together with half the haystack, while INDIGO-Ni removes the haystack, leaving you with the lone needle.
If you are still unconvinced, feel free to ask for a FREE SAMPLE and test out INDIGO-Ni resin.

Additional INDIGO Resins in detail

INDIGO_protein_purification
Fig.2: PureCube 100 INDIGO Ni-Agarose is compatible with 20 mM EDTA and 20 mM DTT. SDS-PAGE of JNK1 expressed in E.coli and purified with PureCube 100 INDIGO Ni-Agarose in the presence of 20 mM EDTA and 20 mM DTT. High yield (>80 mg/ml) and purity were obtained.
INDIGO_protein_purification
Fig.3: PureCube 100 INDIGO Ni-Agarose outperforms competitor products. His-tagged GFP was purified on PureCube 100 INDIGO Ni-Agarose and two leading competitor matrices. Yields obtained with the INDIGO matrix were considerably higher at comparable purity. Buffer conditions: Sodium phosphate buffer pH 7.4, 10 mM DTT, 20 mM EDTA. Imidazole concentrations: Binding step: 10 mM, Wash: 20 mM, Elution: 250 mM.
INDIGO_chromatogram
INDIGO_SDS_Page;protein purification
Fig.4: PureCube 100 INDIGO Ni-Agarose can be re-used multiple times without regeneration. GFP was spiked into E.coli lysates and purified in eight aliquots on the same 1 ml column filled with PureCube 100 INDIGO Ni-Agarose. Between each run, the column was briefly washed with loading buffer containing PBS and 10 mM imidazole. No decrease in performance was observed, even after eight consecutive runs. Left: Chromatogram; Right: SDS-PAGE. M: Marker, L: Lysate, S: Lysate spiked with GFP.

Features

Usage Specific binding and purification of 6x his-tagged proteins
Specifity Affinity to His-tagged proteins
Binding capacity 100 mg/mL
Chelator Stability Stable in buffer containing 20 mM DTT and 20 mM EDTA
pH tolerance pH tolerant from pH 4-9.
Filling quantity Delivered as a 50 % suspension
Bead size 100 μm
Bead Ligand INDIGO-Ni
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 INDIGO-Ni 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 INDIGO-Ni 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 INDIGO-Ni 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.
 C.Regeneration protocol for 100 INDIGO-Ni agarose
 
Recommended after each use. Latest after the 5th one.
 
CV= column volume. E.g. for 1 ml column bed volume use 10 CV = 10 ml of buffer
  1. Wash with 10 x CV of H2O
  2. Wash with 10 x CV of 500 mM NaOH
  3. Wash with 10 x CV of H2O
  4. Wash with 10 x CV Neutralization Buffer (150 mM sodium chloride; 200 mM Na2HPO4, pH 7.0)
  5. Wash with 10 x CV of H2O
  6. Wash with 10 x CV 20% (v/v) Ethanol, 10 mM sodium acetate, pH 6.50 Note: This is the recommended storage buffer

F

protein_purification_resins
The His tag is the most widely used affinity tag due to its small size, low immuno genicity, and versatility under native or denaturing conditions, as well as in presence of detergents and many other additives. Cube Biotech introduces the novel PureCube 100 INDIGO Ni-Agarose, based on high quality crosslinked agarose with 100 µm mean diameter, providing high flow rates in batch and FPLC applications. Using the novel, proprietary INDIGO ligand, purification of his-tagged proteins is possible in the presence of up to 20 mM DTT and 20 mM EDTA (Fig.2) at a protein capacity considerably higher than for comparable competitor products (Fig.3). The novel INDIGO ligand cannot be stripped with EDTA. However, without any regeneration, PureCube 100 INDIGO Ni-Agarose was re-used eight times without reduction in performance (Fig.4). PureCube 100 INDIGO Ni-Agarose is provided as a 50% suspension. To detect His-tagged proteins in Western Blot experiments, Cube Biotech offers the highly specific PentaHis antibody.

Why PureCube 100 INDIGO Ni-Agarose?

  • Stable in 20 mM DTT and 20 mM EDTA, at pH 2-13
  • High yield (up to 80 mg/mL) and purity
  • Compatible with eukaryotic cell culture media
  • Also available as INDIGO Ni-MagBeads

Purity and affinity - superior to our competitors

The purification of proteins always involves the balance act between purity and affinity of your protein. This is especially important when working with the HIS tag. The binding of his tagged proteins to the metal ion of the agarose resin or magnetic beads is based on electric charges. and not distinct peptide sequences. Therefore it tends to impurity as other moleculed in the cell can also be slightly charged similar to a poly his tag.
Our R&D Team however managed to overcome this issue: We developed a new ligand that we named INDIGO, due to its color. Besides its highly superior EDTA and DTT stability (more to that later), its affinity is on par with traditional Ni-Agarose resins, while simultaneously having a highly increased specificity and therefore purity (see figure 1).
High specificity with low expressed proteins
This is especially important when working with low expressed proteins. Low protein amounts lead to highly impure protein purifications with traditional Ni-Agarose as many agarose beads remain uncovered by the protein of interest due to its low abundance. Unspecific binding of unwanted peptides then occurs at these beads that subsequent lead to impurities later on (see figure 1). INDIGO-Ni agarose overcomes this issue, because even at low proteins of interest concentrations in the cell the binding of the his tag to the agarose beads remains highly specific (figure 1).
INDIGO Purification qualities
Fig. 1: Overview of INDIGO-Ni agarose resin's purification properties. The immensly superior puritiy compared to traditional Ni-NTA agarose is worth mentioning. Especially for low expressing proteins. Left side: The performance of our PureCube 100 INDIGO resin. Right side: Performance of Ni-NTA agarose from competitor T.
For this demonstration his tagged GFP was added in known concentrations (see bottom of the blot) to an E.coli cell lysate. This was done to mimic a low protein expression rates with different distinct protein concentrations. As it can be seen both Ni-NTA from competitor T and INDIGO-Ni agarose resin purify his tagged GFP, even at very low concentrations. However the INDIGO concentration is highly superior in purity.
To put it simple: If your protein of interest (POI) is as rare as the famous needle in the haystack, traditional NI-agarose gives you the POI together with half the haystack, while INDIGO-Ni removes the haystack, leaving you with the lone needle.
If you are still unconvinced, feel free to ask for a FREE SAMPLE and test out INDIGO-Ni resin.

Additional INDIGO Resins in detail

INDIGO_protein_purification
Fig.2: PureCube 100 INDIGO Ni-Agarose is compatible with 20 mM EDTA and 20 mM DTT. SDS-PAGE of JNK1 expressed in E.coli and purified with PureCube 100 INDIGO Ni-Agarose in the presence of 20 mM EDTA and 20 mM DTT. High yield (>80 mg/ml) and purity were obtained.
INDIGO_protein_purification
Fig.3: PureCube 100 INDIGO Ni-Agarose outperforms competitor products. His-tagged GFP was purified on PureCube 100 INDIGO Ni-Agarose and two leading competitor matrices. Yields obtained with the INDIGO matrix were considerably higher at comparable purity. Buffer conditions: Sodium phosphate buffer pH 7.4, 10 mM DTT, 20 mM EDTA. Imidazole concentrations: Binding step: 10 mM, Wash: 20 mM, Elution: 250 mM.
INDIGO_chromatogram
INDIGO_SDS_Page;protein purification
Fig.4: PureCube 100 INDIGO Ni-Agarose can be re-used multiple times without regeneration. GFP was spiked into E.coli lysates and purified in eight aliquots on the same 1 ml column filled with PureCube 100 INDIGO Ni-Agarose. Between each run, the column was briefly washed with loading buffer containing PBS and 10 mM imidazole. No decrease in performance was observed, even after eight consecutive runs. Left: Chromatogram; Right: SDS-PAGE. M: Marker, L: Lysate, S: Lysate spiked with GFP.

Features

Usage Specific binding and purification of 6x his-tagged proteins
Specifity Affinity to His-tagged proteins
Binding capacity 100 mg/mL
Chelator Stability Stable in buffer containing 20 mM DTT and 20 mM EDTA
pH tolerance pH tolerant from pH 4-9.
Filling quantity Delivered as a 50 % suspension
Bead size 100 μm
Bead Ligand INDIGO-Ni
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 INDIGO-Ni 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 INDIGO-Ni 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 INDIGO-Ni 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.
 C.Regeneration protocol for 100 INDIGO-Ni agarose
 
Recommended after each use. Latest after the 5th one.
 
CV= column volume. E.g. for 1 ml column bed volume use 10 CV = 10 ml of buffer
  1. Wash with 10 x CV of H2O
  2. Wash with 10 x CV of 500 mM NaOH
  3. Wash with 10 x CV of H2O
  4. Wash with 10 x CV Neutralization Buffer (150 mM sodium chloride; 200 mM Na2HPO4, pH 7.0)
  5. Wash with 10 x CV of H2O
  6. Wash with 10 x CV 20% (v/v) Ethanol, 10 mM sodium acetate, pH 6.50 Note: This is the recommended storage buffer

F

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100 INDIGO Ni-Agarose
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PureCube 100 INDIGO Ni-Agarose PureCube 100 INDIGO Ni-Agarose
50% Agarose of 100 micron average bead size, equipped with a proprietary ligand for His affinity purification. Loaded with nickel (II) sulfate. For more information read:...
Article number: 75103
Sales price: From €84.00 * €105.00 *
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