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 ~40 mg protein per mL of settled beads, as determined by purification of 6xHis-tagged GFP protein from E.coli cleared lysates, and quantified via spectrophotometry.

2. Compatibility

PureCube Ni-NTA MagBeads XL are very stable and can resist the following conditions in most situations: pH 2-4, 100% methanol, 100% ethanol, 8 M urea, 6 M guanidinium hydrochloride, 30% (v/v) acetonitrile.

Fig.1 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 - Regarding Purification capabilities.