PureCube 100 Ni-INDIGO Agarose

Order number: 75103

€150.00*

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Description

PureCube 100 Ni-INDIGO agarose resin is a unique development by Cube Biotech for His-tag protein purifications of the highest quality. Created with the aim in mind to create an agarose resin that can withstand highest concentrations of chelators like EDTA and DTT, which are common in mammalian cell culture buffers. The resulting INDIGO ligand has both DTT and EDTA tolerances of 20 mM each. That is highly superior to other competing resins. Ni-INDIGO is also available as Magnetic Beads or as pre-packed in cartridges / columns.
Feature
Usage Specific binding and purification of 6x His tagged proteins
Specificity Affinity to His tagged proteins
Binding capacity >100 mg/mL
Bead Ligand Ni-INDIGO
Bead size 100 μm
Chelator stability Stable in buffer containing 20 mM DTT and 20 mM EDTA
Filling quantity Delivered as a 50 % suspension
Required equipment
  • Lysis Buffer
  • Wash Buffer
  • Elution Buffer
  • Ice bath
  • Refrigerated centrifuge for 50 mL tube (min 10,000 x g)
  • 50 mL centrifuge tube
  • Micropipettor and Micropipetting tips
  • Disposable gravity flow columns with capped bottom outlet, 2 ml
  • pH meter
  • End-over-end shaker
  • SDS-PAGE buffers, reagents and equipment Optional: Western Blot reagents and equipment

Citations

 ProteinYearAuthor
HisSUMO–kindlin-3 F0 2019 Klapproth S., Bromberger T., Türk C., Krüger M., Moser M.
M protein of SARS-CoV-2 2020 Westberg M., Su Y., Zou X., Ning L., Hurst B., Tarbet B., Lin M.Z.
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.
emST 2021 Weihou G.
C-MET927 and c-MET927-LZ 2021 Uchikawa E., Zhiming C., Guan-Yu X., Xuewu Z. & Xiao-chen B.
several His-tagged proteins 2022 Szuba-Jablonski K., Greig C., Riley D., Italia V., Argue T., Meissner K.E.
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.
several His-tagged proteins 2022 Travis A.
Brazzein 2023 Chua B.N., Guo W.M., Wong H.T., Ow D. S-W., Ho P.L., Koh W., Koay A., Wong F.T.
TccC3hv 2023 Heber S., Borho J., Stadler N., Wondanz F., Irina K., Michaelis J., Papatheodorou P., Barth H., Fellermann M.
His6-MBP-Sec3-1-320 2023 Foltman M., Mendez I., Bech-Serra J.J., de la Torre C., Brace J.L., Weiss E.L., Lucas M., Queralt E., Sanchez-Diaz A.
PcrV 2023 Simonis A., Kreer C., Albus A., Rox K., Yuan B., Holzmann D., Wilms J.A., Zuber S., Kottege L., Winter S., Meyer M., Schmitt K., Gruell H., Theobald S.J., Hellmann A-M., Meyer C., Ercanoglu M.S., Cramer N., Munder A., Hallek M., Rybniker J.
Peptidase Constructs 2023 Tiong E., Koo Y.S., Bi J., Koduru L., Koh W., Lim Y.H., Wong F. T.
QSOX1 2024 Koelbel C., Ruiz Y., Wan Z., Wang S., Ho T., Lake D.
Spike RBD Domain and AchBP 2024 Lina S., Kost V., Miorov V., Sukhov D., Arkhanagelskaya P., Ivanov I., Kudryavtsev D., Siniavin A., Uktin Y., Kasheverov I.
mACE-2 ectodomain 2024 Albert M-C., Uranga-Murillo I., Arias M., De Miguel D., Peña N., Montinaro A., Varanda A. B., Theobald S.J., Areso I., Saggau J., Koch M., Liccardi G., Peltzer N., Rybniker J., Hurtago-Guerrero R., Merino P., Monzón M., Badiola J.J., Reingl-Schwaighöfer R., Sanz-Pamplona R., Cebollada-Solanas A., Megyesfalvi Z., Dome B., Secrier M., Hartmann B., Bergmann M., Pardo J., Walczak H.

Lab Results

INDIGO's key features

INDIGO was developed to solve two problems when it comes to His-tag protein purifications:
  1. The tolerance to certain cell buffer ingredients
  2. The comparatively low purity of His-tag purifications in general
The main idea was to keep the standard purification protocol that was used with e.g. Ni-NTA beads unchanged. Everything remains unchanged, except the agarose beads / magnetic beads.

The chelator ligand that is mostly responsible for the tolerances against chemicals, capacity and purity. Therefore the goal was to develop & introduce a ligand that withstands commonly used reagents like EDTA, DTT and phenanthroline to give the user a huge benefit.
INDIGO's superior DTT and EDTA tolerance
Figure 1: Protein Yield of His-tagged protein compared between INDIGO based beads and competitor products at 20 mM EDTA and 10 mM DTT.
EDTA and DTT resistance of the INDIGO ligand

Protein purification is not limited to simple organisms like E.coli. Also more complex cells like mammalian cells or insect cell lines can be used to express & purify proteins. These systems come with more complex buffer compositions and some of their ingredients may interfere with traditional IMAC ligands. These include:
EDTA is often used in mammalian cell buffers to inhibit any proteases that would decrease the protein yield. But it also strips the NTA and IDA ligands of their nickel ions, thus making the beads useless.
DTT on the other hand can be used to dissolve protein aggregates in the cell lysate that might hinder the access to the protein's His-tag. By reducing nickel, thus making the beads useless.
Phenanthroline as a protease inhbitor is a strong chelator. And therefore behaves similar to EDTA.
To address all these issues, our R&D Team developed our INDIGO ligand that increases to EDTA, DTT and phenanthroline tolerance of a His-tag purification procedure up to 20 mM each.
EDTA tolerance of PureCube Ni-NTA
Figure 2: Elution fractions of a His-tag purification assay using Ni-INDIGO agarose resin with 20 mM EDTA in the purification buffers.
INDIGO's protein purity compared to Ni-NTA & reuseability

As previously mentioned, His-tag protein purification assays can fall behind in terms of purity compared to other affinity tags. Good examples for this are antibody-affinity tags like FLAG or Rho1D4.

On the other hand, FLAG and Rho1D4 purifications, result in very low protein amounts (up to 3-4 mg/ml resin at best) and are better suited for proteins with low abundance.
INDIGO reuseability
Figure 3: PureCube 100 Ni-INDIGO 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 Ni-INDIGO Agarose. Between each run, the column was briefly washed with loading buffer containing PBS and 10 mM imidazole.
His-tag protein purification achieves the highest possible protein yields. In our in-house quality controls for Ni-INDIGO resin we usually get around 100 mg protein per ml resin. The protein used for the quality controls is His-tagged GFP. This Video-Guide shows His-Tag purification using our His-Affinity beads. Furthermore, INDIGO beads can be reused multiple times as demonstrated in figure 3. In addition to that they can be regenerated following this protocol.
INDIGO Compared to other competitors
Figure 4: PureCube Ni-NTA Agarose is robust against oxidation and regenerable. PureCube Ni-NTA Agarose was exposed to 5mM DTT for 1 h (A). After demonstrating that it could still bind GFP (B), the resin was washed, stripped (C), and reloaded with Ni2+ (D) following standard Cube protocol (see Cube Protocols & Datasheets).

Video

Video Guide - How to pack FPLC cartridges


Video Guide - FPLC


Video Guide - Column Chromatography


Video Guide - Batch Spin Chromatography


FAQ

Can I get the datasheet for the 100 INDIGO-Ni agarose resin?

What are the reasons for non specific binding?

Some histidine rich proteins can also bind to nickel. But washing with NaOH after elution of your protein of interest removes unspecific bound proteins from your resin.

I want to use high concentration of EDTA and DTT. Is it possible to use Ni-INDIGO from Cube Biotech?

Yes, of course. Infact Ni-INDIGO was developed with the intention to be highly tolerant towards chelators like EDTA and reducing agents like DTT. Both substances are tolerated up to 20mM.

What is the difference of Ni-INDIGO to Ni-NTA?

As the name suggest the ligand that binds to nickel ions to the purification beads is different. INDIGO binds ions like stronger than NTA an IDA. This results in the increased EDTA and DTT tolerance.

Is protein purification with Ni-INDIGO more complicated than e.g. Ni-NTA

No, it is not. Infact, both of our Ni-NTA beads and their corresponding Ni-INDIGO counterpart share the same protocols. This applies to purifications using native and denaturing conditions.

Can Ni-INDIGO beads be stripped like NTA or IDA beads?

No, this is not possible.

Can I resuse Ni-INDIGO beads or can I regenerate them?

Yes, you can reuse the beads! We recommend to use our INDIGO washing & regeneration protocol at least after every 5th use of the beads.

Note that protocols for e.g. Ni-NTA bead regeneration do NOT apply to INDIGO beads.