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Instructions
for use

NTA-modified Sensor Chips (PDF download)

Product description

Product code Prefix (designates the instrument): SCB, SCBS, SCBN, SPP, SCBI, SPSM, SCH, SPMX, SCR, SCS, SD +
Add: NiP, NiHC30M, NiHC200M, NiHC1000M, NiHC1500M, NiD50L, NiD200M, NiD500L, NiD700M
Example: SCBS NiHC200M
Intended purpose Reversible capture immobilization of His-tagged biomolecules is achieved through complex formation with transition metal ions, preferably Ni2+, which are pre-immobilized on nitrilotriacetic acid (NTA)-modified polycarboxylate sensor chips.
Due to their exceptional binding stability and high capture capacity, NiHC sensor chips are particularly suited for biological systems involving small, tight-binding analytes. Additional recommended applications include the study of biomolecular interactions between proteins and nucleic acids, as well as proteins and peptides.
Storage Store at -20 °C, desiccated over molecular sieve 4A
or at 2–8 °C in physiological buffer.
Related products
  • Buffer kit for NiD and NiHC sensor chips, product code K NI-50I
  • Regeneration kit intensive, product code K RIN-50I

Introduction

NiP, NiDxx or NiHCx sensor chips are coated with a bioinert (poly)carboxylate hydrogel matrix that has been modified with nitrilotriacetic acid (NTA) functionalities. These sensor chips allow the reversible capture immobilization of His-tagged biomolecules through complex formation with transition metal ions, preferably Ni2+. For sufficient binding stability, the His-tag should contain six to ten histidine residues.

The binding affinity of Ni2+-loaded NiP and NiD sensor chips is primarily governed by the typical monovalent interaction between NTA(Ni2+) and His-tags, with dissociation rates (koff) in the range of 10−3 s−1. In contrast, XanTec’s NiHC sensor chips exhibit a multivalent binding character, leading to a significant increase in binding stability of up to three orders of magnitude. Baselines after His-tagged ligand capture show minimal to no drift, with typical koff values of 10-5–10−6 s−1. Due to their exceptionally high binding stability, combined with high ligand capture capacity and ease of regeneration, NiHC sensor chips (particularly NiHC200M and NiHC1500M) are ideally suited for investigating small, tight binders that are difficult to regenerate.

For complete regeneration of NiD and NiHC sensor chips, the (multi)NTA(Ni2+)-His-tag complex can be cleaved by chelating agents such as EDTA or imidazole. When standard regeneration is insufficient, more stringent conditions may be required. In such cases, the Regeneration Kit Intensive is recommended to achieve complete and quantitative regeneration of the NTA sensor chip.

RG Sensor Chips
Fig. 1: Comparison of the affinity distribution plot for a NiHC1000M surface (top) versus an NTA-modified CM dextran hydrogel (bottom). The multivalent binding character of the NiHC sensor chip significantly enhances the binding stability (koff) of the NTA(Ni2+)-His6-tag binding complex.

Additional materials required capturing

Buffer kit for NiD and NiHC sensor chips (product code K NI-50I), Please note that all components of the buffer kit are also available separately.

His-tagged biomolecule (to be provided by the user)

Optional: Regeneration kit intensive (product code K RIN-50I)

Preparations for His-tag capture immobilization

Clean the SPR fluidics

Ensure that the flow system of your SPR equipment is free from any protein contamination, as even small amounts of desorbed protein can accumulate on the charged sensor surface. If necessary, clean the system using either 1 % Tween 20 or, for a more stringent cleaning, 0.5 % SDS for 5 minutes, followed by 50 mM glycine·HCl (pH 9.5) for 10 minutes (both included in the Desorb Kit, product code K D-500ML). The glycine is required to remove residual traces of SDS.

Buffer preparation

Dilute 1 part of 10× HBSTE with 9 parts ultrapure water to prepare the HBSTE running buffer. Ensure the water is 0.22 μm filtered. Mix thoroughly, taking care to avoid introducing air into the buffer. If your system does not include an online degasser, degas the buffer manually before use. Do NOT use HBSTE+, as its higher EDTA concentration can leach Ni2+ ions from the sensor chip surface.

Ligand preparation

In most cases, it is sufficient to dilute the His-tagged ligand in HBSTE running buffer. Suitable concentrations depend on the user requirements but are mostly within the range of 2–200 nM.

Sensor chip

Allow the sealed sensor chip pouch to equilibrate to room temperature to prevent condensation on the chip surface.

After opening the pouch, install the sensor chip according to the instrument manufacturer’s instructions.

Note: XanTec SPR sensor chips, like all nanocoatings, are prone to degradation when exposed to the atmosphere due to reactive oxygen species in the air. To prevent this, unmounted sensor chips should be stored in a closed container under an inert gas atmosphere or in a physiological buffer for short-term storage.

Protocol for His-tag capture immobilization

Procedure Flowrate
[µL/min]		 Injection
time [s]
1 Equilibrate your SPR-system with HBSTE running buffer and mount a compatible XanTec NiD, NiHC or NiP sensor chip. HBSTE was found to give the best results but PBSTE and TBSTE work as well. Small concentrations of EDTA (max. 50 µM) in the running buffer stabilize the assay as they scavenge possible divalent metal ion contaminations.
2 Condition the surface with EDTA regeneration buffer.
Wait until the baseline has stabilized.		 25 3 × 60
3 Inject NiCl2 loading buffer. Depending on the density of chelating groups, a baseline increase by 30–300 RU should be observed. A reference channel should be diverted during this step.
Wait for 5 min or until the baseline has stabilized.		 15 120
4 Inject 2–200 nM His-tagged ligand, preferably in HBSTE running buffer. Immobilization levels can be adjusted by varying the injection time and protein concentration. Start with low concentrations and increase the immobilization level as needed.
For NiD surfaces, keep the immobilization level relatively low, as a certain number of unoccupied NTA(Ni2+) complexes is typically required for stabilization. Note that NiHC surfaces, due to multivalent interactions, exhibit significantly higher binding stability.		 5–25 120–900
5 Wait for 1–5 min and start interaction experiments.
6 Regenerate the sensor chip with 2 pulses of EDTA regeneration buffer. If regeneration outcome is insufficient, add another 60 s injection with 500 mM imidazole and/or 60 s injection of elution buffer pH 12. After complete regeneration, start another capture cycle beginning at step 3. If regeneration remains inadequate, refer to the procedure for intensive regeneration outlined below. 25 2 × 60

Notes

For most biological systems, we recommend using the free NTA surface without Ni2+ loading as a reference because nonspecific binding on Ni2+ loaded NTA surfaces is usually higher than on Ni2+ loaded NTA sensor chips plus captured His-tagged ligand.

Optional: Intensive regeneration

In some situations, EDTA regeneration, even in combination with imidazole and/or elution buffer, may not be sufficient to remove the ligand or nonspecifically bound analyte. If this occurs, we recommend the following procedure:

Procedure for intensive regeneration Flowrate
[µL/min]		 Injection
time [s]
1 Inject 5 M guanidine thiocyanate, 5 mM TCEP. Adjust injection time as required.
Wash with running buffer.		 25 300
2 Inject pepsin solution (1 mg/mL pepsin in 1 M glycine, pH 2.5, freshly prepared). Adjust injection time as required.
Wash with running buffer.		 25 300
3 Inject Conditioning buffer 1. Adjust injection time as required.
Wash with running buffer.		 25 300

Note

The described regeneration procedure is based on the procedure described by Gunnarsson et al. [4] and consistently showed a regeneration effectiveness of more than 99 %. It should be noted that not all regeneration steps have to be applied to all ligands. This should be tested on a case-by-case basis.

Please note that NiHC sensor chips can show a minor initial base line drop after injection of Conditioning buffer 1.

All three additional regeneration solutions are available as part of the Regeneration Kit Intensive.

Ensure that the cooling function of your autosampler is switched off when using 5 M guanidine thiocyanate, as precipitation may occur.

Storage of used sensor chips

For later reuse, sensor chips can be stored either dry or wet under physiological conditions. When handling the sensor chip, avoid touching the top coating with gloves or tweezers. Reuse of planar NiP sensor chips is not recommended due to the lower overall stability and robustness of planar sensor coatings.

Biacore users only: To prevent detachment of the glass chip in the instrument after chips have been stored under buffer or at 100 % humidity, we strongly recommend checking the mechanical stability of the assembly before inserting the chip cartridge into the instrument.

Reichert users only: If the sensor chip is intended for later reuse, use the refractive index matching foil instead of immersion oil when installing the sensor chip for the first time. Oil traces may contaminate the hydrogel top coating after chip removal.

Dry storage
1 Dismount the used sensor chip from your SPR instrument.
2 Rinse the hydrogel surface of the sensor chip carefully with ultrapure water.
3 Dry the sensor chip with a jet of filtered air or nitrogen.
4 Store the sensor chip dry, using a 3A or 4A molecular sieve, in a cold environment (-25 °C) under an inert gas atmosphere in a tightly sealed container. The hydrogel coating should remain stable for several weeks to months.
5 Reinstallation
Equilibrate the sensor chip to room temperature before opening the storage container, then insert the chip according to the instrument manufacturer‘s instructions.
Wet storage
1 Dismount the used sensor chip from your SPR instrument.
2 Rinse the surface of the sensor chip carefully with ultra-pure water. Place the sensor chip in a container filled with sterile filtered, physiological buffer and seal it tightly. For Cytiva sensor chips, 50 mL centrifugation tubes are applicable. Store the sensor chip refrigerated at 2–8 °C.
The sensor coating should be stable for several days to weeks at such conditions. Long-term storage in water is not advised, as this can negatively affect the integrity of the sensor coating.
3 Reinstallation
Remove the sensor chip from the container, preferably using clean tweezers. Rinse with ultra-pure water to remove buffer salts, and carefully dry it using a jet of filtered air or nitrogen. Then, insert the chip according to the instrument manufacturer‘s instructions.

Troubleshooting

Issue Possible solution
Insufficient capture level Increase the concentration of your His-tagged biomolecule.
Make sure that the NTA groups on your NTA sensor chip are loaded with Ni2+ ions.
Use another Ni sensor chip with a higher capture capacity.
Insufficient capture stability Reduce the capture level to approximately one-third of the maximum to achieve optimal capture stability.
If you are using a NiD sensor chip, consider switching to a NiHC sensor chip if applicable.
To covalently couple the ligand, activate your NiHC or NiD sensor chip using EDC/NHS. This approach is particularly useful when regeneration conditions for your biological system do not need to be established.
Insufficient surface regeneration Add a subsequent 60 s injection with 500 mM imidazole and check regeneration efficacy.
If still insufficient, inject elution buffer pH 12 for 60 s and check regeneration efficacy again.
If regeneration is still insufficient, apply the intensive regeneration protocol.

Literature

  1. Nieba, L., Nieba-Axmann, S.E., Persson, A., Hämäläinen, M., Edebratt, F., Hansson, A., Lidholm, J., Magnusson, K., Karlsson, A.F., Plückthun, A. 1996. Biacore Analysis of Histidine-Tagged Proteins Using a Chelating NTA Sensor Chip. Anal. Biochem., 252, 217–228
  2. O‘Shannessy, D. J., O‘Donnell, K.C., Martin, J., Brigham-Burke, M. 1995. Detection and quantitation of hexa-histidine-tagged recombinant proteins on western blots and by a surface plasmon resonance biosensor technique. Anal. Biochem., 229, 119-124
  3. Gershon, P. D., Khilko, S. 1995. Stable chelating linkage for reversible immobilization of oligohistidine tagged proteins in the BIAcore surface plasmon resonance detector. J. Imm. Meth., 183, 65-76
  4. Gunnarsson, A., Stubbs, C. J., Rawlins, P. B., Taylor-Newman, E., Lee, W. C., Geschwindner, S. Dahl, G. (2021). Regenerable Biosensors for Small-Molecule Kinetic Characterization Using SPR. SLAS DISCOVERY: Advancing the Science of Drug Discovery, 26(5), 730-739.

V. 01/25a

For in-vitro use only. Not for use in clinical diagnostic procedures.