Cube Biotech introduces the novel INDIGO-Ni product line for His-tag protein purifications. The novel ligand INDIGO is 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.
|Usage ||Specific binding and purification of 6x His-tagged proteins |
|Specifity ||Affinity to His-tagged proteins |
|Binding capacity ||80 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 |
INDIGO-Ni products from Cube Biotech were successfully used in the following publications:
|HisSUMO–kindlin-3 F0 ||2019 ||Klapproth S., Bromberger T., Türk C., Krüger M., Moser M.1 ||INDIGO-Ni Agarose |
|M protein of SARS-CoV-2 ||2020 ||Westberg M., Su Y., Zou X., Ning L., Hurst B., Tarbet B., Lin M.Z.2 ||INDIGO-Ni Agarose |
|Several His-tagged proteins, ||2020 ||Fellermann M., Huchler C., Fechter L., Kolb T., Wondany F., Mayer D., Michaelis J., Stenger S., Mellert K., Möller P., Barth T.F.E., Fischer S., Barth H. 3 ||INDIGO-Ni Agarose |
|emST ||2021 ||Weihou G.4 ||INDIGO-Ni (agarose resin) |
|C-MET927 and c-MET927-LZ ||2021 ||Uchikawa E., Zhiming C., Guan-Yu X., Xuewu Z. & Xiao-chen B.5 ||100 INDIGO-Ni (agarose resin) |
|several His-tagged proteins ||2022 ||Szuba-Jablonski K., Greig C., Riley D., Italia V., Argue T., Meissner K.E.6 ||INDIGO-Ni (agarose resin) |
|several His-tagged proteins ||2022 ||Struble L.R., Smith A.L., Lutz W.E., Grubbs G., Sagar S., Bayles K.W., Radhakrishnan P., Khurana S., El-Gamal D., Borgstahl G.E.o7 ||INDIGO-Ni (agarose resin) |
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).
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
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.
Additional properties of 100 INDIGO-Ni MagBeads in detail
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.
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- Klapproth, et al. (2019). A kindlin-3–leupaxin–paxillin signaling pathway regulates podosome stability. The Journal of Cell Biology. 10.1083/jcb.201903109.
- Westberg, et al. (2020). Rational design of a new class of protease inhibitors for the potential treatment of coronavirus diseases. bioRxIv. doi.org/10.1101/2020.09.15.275891
- Fellerman, et al. (2020). Clostridial C3 Toxins Enter and Intoxicate Human Dendritic Cells. Toxins. doi.org/10.3390/toxins12090563
- Weihou Guo (2021). Overexpression and isolation of the intermediate state of serotonin transporter from Echinococcus multilocularis –‒ the ER localized HSP complexes of the folding trajectory. PhD, University of Hamburg.
- Uchikawa, E., et al. Structural basis of the activation of c-MET receptor. Nat Commun 12, 4074 (2021). https://doi.org/10.1038/s41467-021-24367-3
- Szuba-Jablonski, K.., et al. Developing ultrabithorax-based sensing platforms. Spi Bios, 4074 (2022). https://doi.org/10.1117/12.2609379
- Struble, L. et al.. (2022). Insect cell expression and purification of recombinant SARS‐COV‐2 spike proteins that demonstrate ACE2 binding. Protein Science. 31. 10.1002/pro.4300.