INDIGO-Ni


Purification Resins
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

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 2-13
Bead size 100 μm
Bead Ligand INDIGO
Reuseable? Yes, up to eight times. See figure 4

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 molecules 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 concentrations 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.

INDIGO Features 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.

Additional properties of 100 INDIGO-Ni MagBeads in detail

MagBeads coupled to GFP
 
MAgBeads GFP JNK1 purification
Fig.5: 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.
If you are still unconvinced, feel free to ask for a FREE SAMPLE and test out INDIGO-Ni resin.
INDIGO-Ni
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PureCube 100 INDIGO Ni-Agarose PureCube 100 INDIGO Ni-Agarose
Cube Biotech's exclusive INDIGO-Ni agarose resin for His Tag protein purification. Up to 100 mg protein / 1 ml resin.
Article number: 75103
Sales price: From €108.00 *
PureCube INDIGO Ni-MagBeads PureCube INDIGO Ni-MagBeads
25% concentrated INDIGO-Ni agarose for His Tag protein purification. EDTA and DTT stable. ~100% purity for your his tagged protein.
Article number: 75201
Sales price: From €69.00 *
PureCube 100 Compact Cartridge Ni-INDIGO PureCube 100 Compact Cartridge Ni-INDIGO
Our exclusive & high quality INDIGO-Ni agarose resin pre-filled in COMPACT FPLC Cartridges / Columns for His Tagged proteins!
Article number: 75302
Sales price: From €50.00 *
PentaHis antibody, BSA-free (0.1 mg) PentaHis antibody, BSA-free (0.1 mg)
Lyophilized. Manufactured by QIAGEN.  
Article number: 40040
Sales price: €448.00 *
PureCube 100 INDIGO Ni-Cartridge PureCube 100 INDIGO Ni-Cartridge
Our exclusive & high quality INDIGO-Ni agarose resin pre-filled in FPLC Cartridges / Columns for your His Tag purifications!
Article number: 75301
Sales price: From €48.00 *
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