| Product code | Prefix (designates the instrument): SCB, SCBS, SCBN, SPP, SCBI, SPSM, SCH, SPMX, SCR, SCS, SD + Add: SPYP, SPYD200M, SPYHC200M Example: SCBS SPYP |
|---|---|
| Intended purpose | Covalent, site-directed immobilization of Spy-tagged proteins on SpyCatcher3™-modified sensor chips in physiological conditions via formation of a stable isopeptide bond. Recommended applications include the investigation of biomolecular interactions involving proteins, nucleic acids and small molecules. |
| Storage | Store at −20 °C desiccated over molecular sieve 4A or at 2–8 °C in physiological buffer. |
| Related products |
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The SPYD, SPYHC, and SPYP sensor chips are coated with a bioinert polycarboxylate matrix, functionalized with the 12.8-kDa SpyCatcher3™ protein. This immobilized SpyCatcher3™ instantly forms highly stable isopeptide bonds with Spy-tagged proteins under physiological conditions, specifically within a pH range of 5 to 8 and a temperature range of 4 to 37 °C.
While site-directed, stable immobilization under physiological buffer conditions is also achievable with the widely-used streptavidin/biotin system (e.g., AviTag™), the SpyCatcher/SpyTag system has two additional advantages. First, the SpyCatcher/SpyTag interaction is truly covalent, resulting in even greater binding stability and facilitating the generation of more reliable experimental data. Second, SpyCatcher is significantly smaller compared to streptavidin, occupying less volume within the sensor coating. This allows for higher ligand capture densities. Combing both benefits, SPYD200M and SPYHC200M chips with its high immobilization capacities for Spy-tagged proteins, are especially well-suited for interaction studies involving short peptides, small molecules, and fragments.
While site-directed, stable immobilization under physiological buffer conditions is achievable with the widely used streptavidin/biotin system (e.g., AviTag™), the SpyCatcher/SpyTag system offers two additional advantages. First, the SpyCatcher/SpyTag interaction is truly covalent, resulting in greater binding stability, which facilitates the generation of reliable experimental data. Second, the SpyCatcher protein is significantly smaller compared to streptavidin, which means it occupies less volume within the sensor coating, allowing for higher ligand capture densities. Combining these benefits, the SPYD200M and SPYHC200M chips, with their high immobilization capacities for Spy-tagged proteins, are especially well-suited for interaction studies involving short peptides, small molecules, and fragments.
SPYP sensor chips, in contrast, offer a unique combination of low immobilization capacity and excellent diffusion characteristics typical of 2D coatings. This makes them particularly useful for all types of protein–protein interaction analyses, especially for interactions characterized by high on- and off-rate kinetics (e.g., weak binders).
XanTec’s sensor chips, modified with SpyCatcher3™, are compatible with SpyTag generations 1–3, ensuring broad applicability for various experimental needs.
Recombinant fusion protein with SpyTag™ binding site (to be provided by the user).
Borate elution buffer (product code: B BELU-50ML): 0.1 M sodium borate, 1 M NaCl, pH 9.0, 50 mL.
10× HBSTE+ buffer (product code B HBSTE+10-500ML): 10× dilution yields 0.01 M HEPES, 0.15 M NaCl, 0.05% Tween 20, 3 mM EDTA, pH 7.4.
Make sure that the flow system of your SPR equipment is free from any protein contamination, because even minor amounts of desorbed protein will concentrate onto the charged sensor surface. If necessary, clean the system with either 1 % Tween 20 or – more stringent – 0.5 % SDS for 5 min followed by 50 mM glycine.HCl (pH 9.5) for 10 min (both part of the Desorb Kit, product code K D-500ML). The glycine is necessary to remove traces of SDS.
Dilute your ligand stock solution directly into the running buffer. Typical protein concentrations are 2–200 nM, but the optimum concentration must be determined experimentally.
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.
| Procedure | Flowrate [µL/min] |
Injection time [s] |
|
|---|---|---|---|
| 1 | Equilibrate your SPR-system with physiological HBSTE+ running buffer and mount a SpyCatcher-modified sensor chip. | ||
| 2 | Condition the surface with Borate elution buffer. Wait until the baseline has stabilized. |
25 | |
| 3 | Inject 2–200 nM Spy-tagged protein, preferably in running buffer. Immobilization levels can be controlled via the injection time and protein concentration. Start with low concentrations and increase immobilization levels as needed. A reference channel should be diverted during the immobilization step. | 10 | 3 × 60 |
| 4 | Optional: Remove loosely physisorbed protein with Borate elution buffer. Wait until the baseline has stabilized. |
25 | 120–900 |
| 5 | If your immobilization level is insufficient, repeat steps 3–4. | 60 | |
| 6 | Start interaction analysis. We recommend beginning with 3–5 consecutive regeneration cycles to improve data quality and stabilize the chip surface. |
It is recommended to start with low ligand concentrations and re-immobilize with higher concentrations as required. This prevents excessive immobilization on the sensor coating. Additional immobilization steps can be conducted during the interaction experiment.
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, SPYD and SPYHC 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 |
|---|---|
| High nonspecific binding | Use a physiological running buffer with Tween like HBSEP or HBSEP+ Add BSA to your running buffer with concentrations of up to 3 %. Increase the salt concentration of the running buffer. |
V. 10/24a
For in-vitro use only. Not for use in clinical diagnostic procedures.