INDIGO Ni-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. Cube Biotech introduces the novel PureCube INDIGO-Ni MagBeads - ferrimagnetic agarose beads coupled to our novel, proprietary INDIGO ligand, loaded with nickel ions. Using the INDIGO ligand, purification of his-tagged proteins is possible in the presence of up to 20 mM DTT and 20 mM EDTA (Fig.1) at high protein capacity. Please note that the novel INDIGO ligand cannot be stripped with EDTA. PureCube INDIGO-Ni MagBeads are delivered as a 25% suspension.

Why PureCube INDIGO-Ni MagBeads?

Superior to our competitors - in both Purity and affinity

The his tag is based on electric charges and not a distinct peptide sequence. Therefore impurities can occur because other proteins can have a slightly similar surface composition. This is especially a problem when purifying a low expression proteins.
However Our R&D Team found a solution to this issue: We developed INDIGO, a new ligand that we named this way due to its color. Its especialy superior in two features compared to traditional agarose resins: superior EDTA and DTT stability. Additionally its protein affinity is on par with traditional Ni-Agarose resins, while at the same time having a highly increased specificity and therefore purification assays result in purer protein as with comparable Ni-NTA (see figure 1).
Low expression proteins: No longer a challenge
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 the properties INDIGO-Ni agarose resin. As the gels presents, the purity of 100 INDIGO resin as far superior to competitor T's Ni-NTA. The shown blot as mimics a low expression protein. 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 distinct concentrations to an E.coli cell lysate to mimic a low expression protein with controlled protein concentrations. As it can be seen both tested agarose resins managed to purify the his tagged GFP. 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 properties of 100 INDIGO-Ni MagBeads in detail

MagBeads coupled to GFP
 
MAgBeads GFP JNK1 purification
Fig.2: Using PureCube INDIGO-Ni MagBeads, his-tagged proteins can be purified at high yield and purity even in the presence of 10 mM DTT and 20 mM EDTA. 15 mL E.coli lysate containing recombinant green fluorescent protein GFP (left) or human c-Jun N-terminal protein kinase 1 JNK1 (right) were applied to 250 µL PureCube INDIGO-Ni MagBeads each. Standard buffers containing 10 mM DTT and 20 mM EDTA were used throughout the purification procedure. Yields of 89 mg GFP and 48 mg JNK1 per mL MagBeads were obtained. MW: Molecular weight marker. L: Cleared lysate, FT: flow through, W: wash; E1-E3: elution fractions 1-3.

Features

Usage Specific binding and purification of his-tagged proteins
Specifity Affinity to His-tagged proteins
High binding capacity 100 mg/mL settled beads
Tolerances
 
  • pH tolerant from pH 4-9
  • Providing stability in 20 mM EDTA and 20 mM DTT.
Filling quantity Delivered as a 25 % suspension
Bead size 25 μm
Bead Ligand INDIGO-Ni
Required equipment
 

Applications

   
A.Protein purification protocol for nactive 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 Native Lysis Buffer supplemented with 1 mg/mL lysozyme.
  3. Add 6 U Benzonase® (3 units/mL bacterial culture) to the lysate to reduce viscosity caused by genomic DNA.
  4. ncubate for 30 min on ice, if necessary. Otherwise, incubating at room temperature (20-25 °C) may be more efficient.
  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 INDIGO-Ni MagBeads by vortexing. Transfer 40 μL of the 25 % magnetic bead suspension into a conical microcentrifuge tube. Note: Depending on the protein expression rate, the quantity of magnetic bead suspension can be adjusted from 2-200 μL.
  7. Add 500 μL Native 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. Tip: Briefly centrifuge the sample before placing it on the magnetic separator in order to collect liquid from the lid.
  10. Remove the tube from the magnet. Add 500 μL Native Wash Buffer and mix by vortexing. Place the tube again on the magnetic microtube stand and allow the beads to separate. Remove the supernatant.
  11. Repeat step 10 twice.
  12. Elute the His-tagged protein using 100 μL Native Elution Buffer. Note: Depending on the protein expression rate and desired protein concentration, the elution volume can be adjusted from 25 to 500 μL.
  13. Repeat step 12. Collect each elution fraction in a separate tube and determine the protein concentration of each fraction.
  14. 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.
  15. Optional: Perform Western Blot experiment using PentaHis Antibody.
   
B.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.
  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. Pipet 1 mL of the cleared lysate into a conical microcentrifuge tube.
  6. Resuspend the PureCube INDIGO-Ni MagBeads by vortexing. Transfer 40 μL of the 25 % magnetic beads suspension onto the lysate. Note: Depending on the protein expression rate and desired protein concentration, the elution volume can be adjusted from 25 to 500 μL.
  7. Incubate the lysate-magnetic bead mixture at room temperature for 1 h on an end-over-end shaker.
  8. Place the tube on the magnetic microtube stand until the beads separate and remove the supernatant. Tip: Briefly centrifuge the sample before placing it on the magnetic separator in order to collect liquid from the lid.
  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.
  10. Repeat step 9 twice.
  11. Elute the His-tagged protein using 100 μL Denaturing Elution Buffer. Note: Depending on the protein expression rate and desired protein concentration, the elution volume can be adjusted from 25 to 500 μL.
  12. Repeat step 11. Collect each elution fraction in a separate tube and determine the protein concentration of each fraction.
  13. Analyze all fractions by SDS-PAGE.
  14. Optional: Perform Western Blot experiment using PentaHis Antibody.Tip: If the target protein is acid-labile, elution can be performed with 250-500 mM imidazole.
  15. 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..
  16. Optional: Perform Western Blot experiment using PentaHis Antibody.
 C.Regeneration protocol for INDIGO-Ni MagBeads
 
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
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. Cube Biotech introduces the novel PureCube INDIGO-Ni MagBeads - ferrimagnetic agarose beads coupled to our novel, proprietary INDIGO ligand, loaded with nickel ions. Using the INDIGO ligand, purification of his-tagged proteins is possible in the presence of up to 20 mM DTT and 20 mM EDTA (Fig.1) at high protein capacity. Please note that the novel INDIGO ligand cannot be stripped with EDTA. PureCube INDIGO-Ni MagBeads are delivered as a 25% suspension.

Why PureCube INDIGO-Ni MagBeads?

Superior to our competitors - in both Purity and affinity

The his tag is based on electric charges and not a distinct peptide sequence. Therefore impurities can occur because other proteins can have a slightly similar surface composition. This is especially a problem when purifying a low expression proteins.
However Our R&D Team found a solution to this issue: We developed INDIGO, a new ligand that we named this way due to its color. Its especialy superior in two features compared to traditional agarose resins: superior EDTA and DTT stability. Additionally its protein affinity is on par with traditional Ni-Agarose resins, while at the same time having a highly increased specificity and therefore purification assays result in purer protein as with comparable Ni-NTA (see figure 1).
Low expression proteins: No longer a challenge
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 the properties INDIGO-Ni agarose resin. As the gels presents, the purity of 100 INDIGO resin as far superior to competitor T's Ni-NTA. The shown blot as mimics a low expression protein. 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 distinct concentrations to an E.coli cell lysate to mimic a low expression protein with controlled protein concentrations. As it can be seen both tested agarose resins managed to purify the his tagged GFP. 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 properties of 100 INDIGO-Ni MagBeads in detail

MagBeads coupled to GFP
 
MAgBeads GFP JNK1 purification
Fig.2: Using PureCube INDIGO-Ni MagBeads, his-tagged proteins can be purified at high yield and purity even in the presence of 10 mM DTT and 20 mM EDTA. 15 mL E.coli lysate containing recombinant green fluorescent protein GFP (left) or human c-Jun N-terminal protein kinase 1 JNK1 (right) were applied to 250 µL PureCube INDIGO-Ni MagBeads each. Standard buffers containing 10 mM DTT and 20 mM EDTA were used throughout the purification procedure. Yields of 89 mg GFP and 48 mg JNK1 per mL MagBeads were obtained. MW: Molecular weight marker. L: Cleared lysate, FT: flow through, W: wash; E1-E3: elution fractions 1-3.

Features

Usage Specific binding and purification of his-tagged proteins
Specifity Affinity to His-tagged proteins
High binding capacity 100 mg/mL settled beads
Tolerances
 
  • pH tolerant from pH 4-9
  • Providing stability in 20 mM EDTA and 20 mM DTT.
Filling quantity Delivered as a 25 % suspension
Bead size 25 μm
Bead Ligand INDIGO-Ni
Required equipment
 

Applications

   
A.Protein purification protocol for nactive 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 Native Lysis Buffer supplemented with 1 mg/mL lysozyme.
  3. Add 6 U Benzonase® (3 units/mL bacterial culture) to the lysate to reduce viscosity caused by genomic DNA.
  4. ncubate for 30 min on ice, if necessary. Otherwise, incubating at room temperature (20-25 °C) may be more efficient.
  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 INDIGO-Ni MagBeads by vortexing. Transfer 40 μL of the 25 % magnetic bead suspension into a conical microcentrifuge tube. Note: Depending on the protein expression rate, the quantity of magnetic bead suspension can be adjusted from 2-200 μL.
  7. Add 500 μL Native 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. Tip: Briefly centrifuge the sample before placing it on the magnetic separator in order to collect liquid from the lid.
  10. Remove the tube from the magnet. Add 500 μL Native Wash Buffer and mix by vortexing. Place the tube again on the magnetic microtube stand and allow the beads to separate. Remove the supernatant.
  11. Repeat step 10 twice.
  12. Elute the His-tagged protein using 100 μL Native Elution Buffer. Note: Depending on the protein expression rate and desired protein concentration, the elution volume can be adjusted from 25 to 500 μL.
  13. Repeat step 12. Collect each elution fraction in a separate tube and determine the protein concentration of each fraction.
  14. 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.
  15. Optional: Perform Western Blot experiment using PentaHis Antibody.
   
B.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.
  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. Pipet 1 mL of the cleared lysate into a conical microcentrifuge tube.
  6. Resuspend the PureCube INDIGO-Ni MagBeads by vortexing. Transfer 40 μL of the 25 % magnetic beads suspension onto the lysate. Note: Depending on the protein expression rate and desired protein concentration, the elution volume can be adjusted from 25 to 500 μL.
  7. Incubate the lysate-magnetic bead mixture at room temperature for 1 h on an end-over-end shaker.
  8. Place the tube on the magnetic microtube stand until the beads separate and remove the supernatant. Tip: Briefly centrifuge the sample before placing it on the magnetic separator in order to collect liquid from the lid.
  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.
  10. Repeat step 9 twice.
  11. Elute the His-tagged protein using 100 μL Denaturing Elution Buffer. Note: Depending on the protein expression rate and desired protein concentration, the elution volume can be adjusted from 25 to 500 μL.
  12. Repeat step 11. Collect each elution fraction in a separate tube and determine the protein concentration of each fraction.
  13. Analyze all fractions by SDS-PAGE.
  14. Optional: Perform Western Blot experiment using PentaHis Antibody.Tip: If the target protein is acid-labile, elution can be performed with 250-500 mM imidazole.
  15. 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..
  16. Optional: Perform Western Blot experiment using PentaHis Antibody.
 C.Regeneration protocol for INDIGO-Ni MagBeads
 
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
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PureCube INDIGO Ni-MagBeads PureCube INDIGO Ni-MagBeads
Article number: 75201
INDIGO Ni-MagBeads
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PureCube INDIGO Ni-MagBeads PureCube INDIGO Ni-MagBeads
1 ml 25% magnetic beads equipped with a proprietary ligand for His affinity purification, loaded with nickel (II) sulfate For more information read here: https://cube-biotech.com/products/magnetic-beads/his-affinity-magbeads/#
Article number: 75201
Sales price: From €67.00 *
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