| Product code | Prefix (designates the instrument): SCB, SCBS, SCBN, SPP, SCBI, SPSM, SCH, SPMX, SCR, SCS, SD Add: HLC30M, HLC200M, HLC1000M, HLC1500M Example: SCBS HLC1000M |
|---|---|
| Intended purpose | HLC sensor chips are a charge-reduced version of XanTec’s HC sensor chips. The low charge of the highly hydrated sensor chip matrix effectively minimizes electrostatic nonspecific interactions, especially with positively charged species. This can improve SPR data quality in complex sample matrices, such as serum and fermentation broths. Similar to their polycarboxylate original, HLC sensor chips are designed for covalent ligand immobilization using EDC/(sulfo-)NHS chemistry. For successful immobilization, the ligand must contain at least one reactive primary amine, thiol, or aldehyde group. Recommended applications include the investigation of biomolecular interactions involving proteins, nucleic acids, and small molecules that present an exceptional challenge due to nonspecific binding. |
| Storage | Store at -20 °C, desiccated over molecular sieve 4A or at 2–8 °C in physiological buffer. |
| Related products |
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XanTec’s HLC sensor chips are based on a 3D hydrogel matrix consisting of flexible polycarboxylate chains (HC) grafted onto a hydrophilic adhesion promoter on a gold support. Compared to the related HC sensor chips, a significant portion (about two-thirds) of the HLC’s carboxyl functionalities has been neutralized. While this modification reduces the immobilization capacity, it minimizes non-specific binding (NSB) caused by electrostatic interactions. Typical applications that benefit from HLC chip coatings involve analytes with multiple positive charges, which often show high non-specific interactions with standard CMD and HC sensor surfaces.
In this product information, we describe covalent ligand immobilization using EDC/(sulfo-)NHS chemistry. A prerequisite for successful amine coupling is the presence of at least one primary amine group on the ligand. While this is unproblematic for most proteins, small molecules, carbohydrates, or oligonucleotides may require additional functionalization.
Sufficient electrostatic preconcentration is essential for successful protein immobilization; therefore, preconcentration scouting of the ligand (see protocol below) should be performed before the actual immobilization. Small molecules, on the other hand, can freely diffuse into the sensor matrix and do not require electrostatic preconcentration for efficient immobilization.
We recommend using HLC sensor chips with either the XanTec Amine Coupling Kit AS or Amine Coupling Kit AN. While standard NHS is effective for immobilizing most proteins with an isoelectric point (pI) ≥ 5, achieving sufficient electrostatic preconcentration on HLC sensor chips can be challenging if the ligand is more acidic. In such cases, the sulfo-NHS-containing Amine Coupling Kit AS provides a viable alternative. The choice between these two kits will depend on the specific characteristics of the ligand and should be evaluated on a case-by-case basis.
Amine coupling kit AN, NHS (product code K AN-50I)
or Amine coupling kit AS, sulfo-NHS (product code K AS-50I)
Coupling buffer (dependent on the pI of the ligand):
Acetate buffer pH 4.0 (product code B A40-50ML): 5 mM sodium acetate, pH 4.0, 50 mL
or Acetate buffer pH 4.5 (product code B A45-50ML): 5 mM sodium acetate, pH 4.5, 50 mL
or Acetate buffer pH 5.0 (product code B A50-50ML): 5 mM sodium acetate, pH 5.0, 50 mL
or Acetate buffer pH 5.5 (product code B A55-50ML): 5 mM sodium acetate, pH 5.5, 50 mL
or Maleate buffer pH 6.0 (product code B M60-50ML): 2.5 mM sodium maleate, pH 6.0, 50 mL
Ligand bearing reactive amino groups (to be provided by the user)
Even high-purity EDC·HCl often contains impurities such as diamines, which can neutralize negative surface charges and even quench the (sulfo-)NHS esters on the activated chip surface. Such artifacts can significantly decrease the immobilization yield, especially at high EDC concentrations, and in the worst-case scenario, the deactivation can be quantitative. Other components in the activation mixture may also introduce trace contaminants that interfere with the process.
The EDC·HCl provided by XanTec has undergone purification and testing to confirm its suitability for SPR applications. It is important to note that the performance of the EDC has been evaluated only up to the designated maximum concentrations. For HC coatings, this limit is approximately 1 % w/v EDC (52 mM) for NHS and 5 % w/v (260 mM) for sulfo-NHS. For CMD coatings, the limit is around 5 % w/v EDC (260 mM) for both NHS and sulfo-NHS. These concentrations are typically sufficient to achieve high coupling yields and immobilization densities with proteins. Particularly for HC coatings, higher EDC concentrations may result in significantly reduced immobilization yields.
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.
In some cases, the ligand solution may contain sodium azide or amine-containing buffers, such as Tris. Both components can react with (sulfo-)NHS esters and negatively impact immobilization efficiency. Therefore, they should be removed from the ligand solution using a desalting procedure before immobilization. Generally, it is advisable to desalt into an azide- and amine-free, pH-neutral buffer with low ionic strength. Desalting directly into a preconcentration buffer can enhance the preconcentration effect after dilution but may sometimes lead to protein loss due to aggregation.
Use concentrated ligand stock solutions (≥1 mg/mL) to ensure that the final ligand solution in the coupling buffer is less affected by the pH and ionic strength of the stock solution.
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.
Aqueous EDC solutions are susceptible to hydrolysis. Therefore, small aliquots of Activation Buffer should be frozen, and the corresponding amount of solid EDC·HCl should be dissolved immediately in the buffer before use. Because EDC is sensitive to humidity, the reagent bottle should be stored at -20 °C or lower in a desiccated container, brought to room temperature before opening, and exposed to air for as short a time as possible, ideally under an inert gas atmosphere.
Alternatively, solid EDC·HCl can be dissolved in ultrapure water to prepare a 10 % EDC solution (521 mM), which can then be divided into separate aliquots for storage at -20 °C or lower. These aliquots remain stable for up to two months when kept frozen. Immediately before use, thaw a frozen aliquot to room temperature, gently shake it, and combine it with the defrosted Activation Buffer (S). This mixture can then be used for activating the sensor chip.
If the pI of your protein is known, a coupling buffer with a pH 0.5–1.0 units below the pI of the ligand is recommended for efficient immobilization. Protein concentrations of 10–100 µg/mL are typically sufficient for efficient covalent coupling.
If the pI of your protein is unknown, consider performing an electrostatic preconcentration scouting. In this case, dilute your protein stock solution into different coupling buffers to achieve final protein concentrations of 5–25 µg/mL. Start at pH 6.0 and decrease the pH in increments of 0.5 until reaching pH 4.0.
Please note that due to the reduced charge, the electrostatic preconcentration effect is diminished to approximately one-third of the preconcentration capacity of the corresponding HC sensor chip.
| Procedure for electrostatic preconcentration scouting | Flowrate [µL/min] |
Injection time [s] |
|
|---|---|---|---|
| 1 | Equilibrate your SPR-system with physiological running buffer. Mount a compatible XanTec sensor chip. |
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| 2 | Condition the surface with Borate elution buffer. Wait until the baseline has stabilized. | 25 | 3 × 60 |
| 3 | Inject your protein (5–25 µg/mL) in Coupling buffer. Start at pH 6.0 (maleate coupling buffer). After protein injection, wait for 60 s, then inject the next protein solution at a pH 0.5 units lower than the previous solution. Repeat until you reach pH 4.0. Select the highest pH value that allows a sufficiently high pre-concentration effect. |
10 | 600 |
| 4 | Inject Borate elution buffer. Wait until the baseline has stabilized. | 25 | 60 |
| Procedure | Flowrate [µL/min] |
Injection time [s] |
|
|---|---|---|---|
| 1 | Equilibrate your SPR-system with water as running buffer and mount a compatible XanTec sensor chip. | ||
| 2 | Condition the surface with Borate elution buffer. Wait until the baseline has stabilized. | 25 | 3 × 60 |
| 3 | Mix solid EDC with Activation Buffer (S). Filter the EDC/Activation Buffer solution through a 0.45 µm filter and inject. Recommended EDC·HCl concentration range: HLC chip (NHS): 0.1–1.0 % HLC chip (sulfo-NHS): 0.5–5.0 % |
15 | 600 |
| 4 | Wash briefly with water and inject the protein solution in a suitable Coupling Buffer. Protein concentrations of 10–100 µg/mL are recommended | 15 | 600 |
| 5 | Inject Ethanolamine quenching buffer. | 15 | 600 |
| 6 | Optional: Remove loosely physisorbed protein with Borate elution buffer. | 25 | 3 × 60 |
| 7 | Switch to physiological running buffer and wait until the SPR-signal has stabilized. | ||
| 8 | Start interaction analysis. We recommend beginning with 3–5 consecutive regeneration cycles to improve data quality and stabilize the chip surface. |
Physiological running buffer can be used instead of water during immobilization, but dispersion effects between the protein coupling solution and the running buffer may slightly reduce the immobilization yield.
Avoid amine- or azide- containing buffers.
Higher immobilization yields can be achieved by lowering the ionic strength of the protein coupling buffer, increasing the protein concentration, raising the EDC concentration, or extending the protein contact time.
Avoid prolonged incubation of the sensor chip in water, as this can negatively affect the integrity of the sensor coating over time. Instead, use a physiological buffer for storage.
The selection of a suitable regeneration buffer is crucial when performing binding studies in which the analyte does not dissociate completely within an adequate period of time. In such cases, the analyte must be removed manually through a regeneration procedure. The goal is to ensure complete analyte removal without reducing ligand activity. Since the specific binding between the ligand and analyte is driven by a unique – and, in most cases, unknown – combination of physical forces, the regeneration conditions must be determined empirically.
Experience has shown that short pulses of 10–20 mM H3PO4 or 10 mM Glycine-HCl at pH 1.5–2.5 (part of Regeneration Scouting Kit 1, product code K RK1-50I) are often sufficient to achieve quantitative regeneration. However, some receptor-ligand pairs may require different conditions for successful regeneration. Occasionally, the interaction between two binding partners is so strong that binding becomes practically irreversible. In such cases, kinetic titration or capture immobilization of the ligand are promising strategies.
Andersson has proposed an innovative algorithm to streamline the otherwise time-consuming process of identifying optimal regeneration conditions [2][3]. His approach involves systematically combining six different regeneration cocktails. The composition of these cocktails, which Xantec distributes as the Regeneration Scouting Kit 2 (product code K RK2-50I), is outlined in the table below:
| Stock solution | Product code | Composition |
|---|---|---|
| Acidic | B RCA-50ML | 37.5 mM Oxalic acid, 37.5 mM H3PO4, 37.5 mM formic acid, 37.5 mM malonic acid pH 5.0 |
| Alkaline | B RCB-50ML | 0.2 M Ethanolamine, 0.2 M Na3PO4, 0.2 M Piperazine, 0.2 M Glycine pH 9.0 |
| Chaotropic | B RCI-50ML | 0.46 M KSCN, 1.83 M MgCl2, 0.92 M Urea, 1.83 M Guanidine·HCl |
| Non-polar, water-soluble | B RCS-50ML | 20 % (v/v) DMSO, 20 % (v/v) formamide, 20 % (v/v) ethanol, 20 % (v/v) acetonitrile, 20 % (v/v) 1-butanol |
| Detergent | B RCD-50ML | 0.3 % (w/v) CHAPS, 0.3 % (w/v) Zwittergent 3-12, 0.3 % (v/v) Tween 80, 0.3 % (v/v) Tween 20, 0.3 % (v/v) Triton X-100 |
| Chelating | B RCC-50ML | 0.02 M disodium EDTA |
| Procedure for regeneration screening and optimization | |
|---|---|
| 1 | Prepare the first regeneration cocktail by mixing one part of one of the six stock solutions with two parts of water. |
| 2 | Inject the analyte until equilibrium is reached. |
| 3 | Screening: Inject the first regeneration cocktail and measure its effect as a percentage of the analyte removed from the sensor chip. No effect corresponds to 0 % regeneration, while complete removal equals 100 % regeneration. If the regeneration efficiency is below 10 %, proceed to inject the next regeneration cocktail (1 part stock solution, 2 parts water). If the analyte level drops below 67 % of the original value, inject new analyte and re-saturate the surface. Repeat until all cocktails have been evaluated. The regeneration efficacy Re is calculated by the following formula: Re= (analyte loss)/(analyte level) × 100 % |
| 4 | Optimization: Identify the two to three regeneration cocktails with the highest Re and recombine them using a 2D (two best regeneration cocktails) or 3D (three best regeneration cocktails) experimental mixture design. Add water if the new cocktails do not reach 100 % volume. Re-evaluate the Re of the new regeneration solutions. |
| 5 | If regeneration remains insufficient, follow the trends observed in the previous optimization experiment and iterate until regeneration is satisfactory. Note: Repeated short pulses of the regeneration solution are generally more effective than increasing the injection time. |
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.
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, potentially causing irreversible damage to the immobilized ligand.
| 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 | Optional: Carefully remove excess water from the edge of the hydrogel coating using a pipette. Place a droplet (30 µL for SCB and SCBS) of XanTec stabilization buffer onto the wet chip surface and allow it to spread, ensuring it covers the entire surface. Let it dry for approximately 60 minutes in a desiccator with desiccant (4A molecular sieve). This step helps prevent denaturation of the immobilized ligand and prolongs the shelf-life of the sensor chip. |
| 4 | Dry the sensor chip with a jet of filtered air or nitrogen. |
| 5 | 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 stability of the sensor chip depends on the stability of the immobilized ligand. The underlying hydrogel coating should remain stable for several weeks to months. |
| 6 | Reinstallation No protective top coating: Equilibrate the sensor chip to room temperature before opening the storage container, then insert the chip according to the instrument manufacturer‘s instructions. Protective top coating applied: Equilibrate the sensor chip to room temperature before opening the storage container. Immerse the chip in physiological buffer for 10 minutes to remove the protective layer from the hydrogel coating. Rinse gently with ultra-pure water and carefully dry it using a stream of filtered air or nitrogen. |
| Wet storage | |
|---|---|
| 1 | Dismount the used sensor chip from your SPR instrument. |
| 2 | Rinse the hydrogel 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 stability of the sensor coating mainly depends on the stability of the immobilized ligand. The underlying hydrogel 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 stream of filtered air or nitrogen. Then, insert the chip according to the instrument manufacturer‘s instructions. |
| Issue | Possible solution |
|---|---|
| Insufficient electrostatic preconcentration | Perform electrostatic preconcentration screening to identify optimal preconcentration conditions. Desalt the protein directly into Coupling Buffer to remove potential salt contaminants and lower the ionic strength of the buffer. If the ligand is too acidic (pI < 3.0) and does not preconcentrate on HLC sensor chips, consider using alternative coupling methods. If an unusually high signal increase is observed after EDC/NHS activation, impure EDC might be the cause. Replace the EDC with a fresh vial or try using a different brand. Decrease the ionic strength of the immobilization buffer. Switch to Activation Buffer S, which contains sulfo-NHS, as an alternative to the standard Activation Buffer. Lower the EDC concentration to minimize potential side reactions. |
| Insufficient protein immobilization level | Ensure optimal electrostatic preconcentration conditions are used. Remove interfering components from your protein stock solution, such as azide or amine-containing buffers like Tris. Multiple desalting steps may be necessary for complete removal. Increase the EDC concentration, if necessary, to enhance coupling efficiency. Extend the protein contact time to improve immobilization yields. Increase the protein concentration for better preconcentration and coupling. |
| Insufficient ligand activity | Check ligand integrity in your stock solution and immobilization buffer with respect to activity, aggregation, and biological contamination. Not all proteins tolerate low pH or low ionic strength. Lower the EDC concentration to avoid ligand crosslinking. Reduce the overall immobilization level to minimize ligand crowding on the sensor surface. If your protein is sensitive to acidic pH, increase the pH of the Coupling Buffer. If physiological conditions are required, consider an alternative immobilization strategy. If the ligand couples at its active site, consider using alternative coupling methods. |
V. 10/24a
For in-vitro use only. Not for use in clinical diagnostic procedures.