1.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).

2.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).

1.  Protein Binding Capacity

The protein binding capacity is up to 80 mg/mL, as determined by purification of 6xHis-tagged GFP protein from E.coli cleared lysates, and quantified via spectrophotometry.

2.  Compatibility

PureCube 100 INDIGO Ni-Agarose is very stable and can resist the following conditions in most situations: buffers at pH 4-13, 100% methanol, 100% ethanol, 8 M urea, 6 M guanidinium hydrochloride, 30% (v/v) acetonitrile, 20 mM DTT, 20 mM EDTA.

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.

 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.

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.

 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.