PureCube 100 INDIGO Ni-Agarose
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.1) at a protein capacity considerably higher than for comparable competitor products (Fig.2). 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.3). 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
Fig.1: 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.2: 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.3: 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.