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Product information "HisCube - Ni-INDIGO His-Tag Protein Purification MIDI Kit"
The HisCube His-tag protein purification MIDI Kit is our way to supply your laboratory with a convenient method for high-quality small scale His-tag protein purifications. We put all of our experience in His-tag purifications into this Kit to provide a protocol that is easy to follow and does not contain unnecessary equipment.
The next task that our R&D team set up for themselves was to improve His-tag protein purifications. Their solution was our in-house developed ligand INDIGO. INDIGO replaces NTA or IDA that is used in traditional His-tag protein purification beads but comes with additional advantages. INDIGO tolerates up to 20 mM EDTA and DTT in the purification buffers. In contrast: NTA tolerates 10 mM DTT and 1 mM EDTA.
But what are the advantages of EDTA and DTT in the buffers?
EDTA is a chelator that is often used in eukaryotic cell buffers because it has protease inhibiting functions.
DTT can dissolve protein aggregates that could potentially hinder the access of the purification beads to the His tagged proteins.
But thanks to INDIGO both substances can be used now. Therefore they are included in the Kit.
|Contents of the Kit:|
|2 x 50 ml Binding Buffer |
2 x 50 ml Wash Buffer
1 x 50 ml Elution Buffer
1 x 10 ml Ni-INDIGO agarose resin
5 x MIDI Spin Columns
1 x 500 mM DTT (add 1,016 mL ddH2O)
1 x 500 mM EDTA (add 0.866 mL ddH2O)
Print-out of the protocol
Print-out of the fast protocol
|Usage||Specific binding and purification of 6x his-tagged proteins|
|Specifity||Affinity to His-tagged proteins|
|Binding capacity||>100 mg/mL|
|Bead size||100 μm|
|Chelator stability||Stable in buffer containing 20 mM DTT and 20 mM EDTA|
|MIDI centriduge / drip columns|
|Filter pore size||0.1-0.2µm low binding PVDF|
|Max. volume||20 mL|
|Max. g force||1,500-2,000 x g (swing-bucket rotor)|
|Min. g force||100-200 x g for 1-10 min|
Figure 1: Westernblot that compared the purification results of His tagged GFP by either using Ni-INDIGO beads or Ni-NTA beads. The Ni-INDIGO beads were produced by Cube Biotech, while the Ni-NTA were manufactured by competitor T.
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