| Product code | Prefix (designates the instrument): SCB, SCBS, SCBN, SPP, SCBI, SPSM, SCH, SPMX, SCR, SCS, SD + Add: SAP, SAHC30M, SAHC200M, SAHC1500M, SAD50L, SAD50M, SAD200L, SAD200M, SAD700L Example: SCBS SAHC200M |
|---|---|
| Intended purpose | Capture immobilization of biotinylated biomolecules under physiological conditions with exceptionally high affinity. XanTec’s streptavidin-modified sensor chips offer an efficient alternative to covalent immobilization, making it ideal for ligands that are challenging to immobilize or sensitive to covalent coupling. Recommended applications include the investigation of biomolecular kinetics involving proteins, peptides and nucleic acids. |
| 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 SAD, SAHC, and SAP sensor chips are coated with a bioinert polycarboxylate matrix, pre-functionalized with a recombinant 52 kDa streptavidin tetramer. This immobilized streptavidin effectively captures biotinylated biomolecules—such as biotinylated proteins, nucleic acids, and peptides—with exceptionally high affinity under physiological conditions.
Streptavidin-modified sensor chips are a popular alternative to conventional amine coupling, particularly when standard amine chemistry poses challenges for ligand immobilization. These challenges often occur when the ligand is too acidic to efficiently pre-concentrate on a polycarboxylate surface or is unstable during the preconcentration process. Streptavidin-functionalized surfaces offer several key advantages, including enhanced control over immobilization levels and the ability to capture ligands in an oriented manner using site-directed biotinylation strategies, such as AviTag™.
Moreover, the streptavidin-biotin complex exhibits exceptional chemical stability, making it highly resistant to most common regeneration protocols. This durability enhances the versatility of streptavidin-modified chips, making them suitable for a wide range of experimental conditions and applications.
XanTec offers a range of streptavidin-modified sensor chips, specifically designed to meet the needs of surface plasmon resonance (SPR) applications. The 2D SAP sensor chips provide superior diffusion characteristics, though they come with lower capture capacities. This makes them an ideal choice for demanding biomolecular interaction analyses, particularly with bulkier analytes and weak binders that exhibit fast on- and off-rates. On the other hand, SAD700L sensor chips support higher immobilization levels, allowing for up to ≈7000 µRIU of immobilization for medium-sized proteins (50–70 kDa). This makes them particularly well-suited for applications involving smaller analytes.
Biotinylated biomolecule (to be provided by the user)
Conditioning buffer 1 (product code B C1-50ML): 50 mM NaOH, 1 M NaCl
Optional: biotin blocking solution (to be provided by the user): 50 µM of a water-soluble biotin-derivate like biocytin or biotin-PEO3-amine in running buffer. Free biotin is only poorly soluble in aqueous solutions and therefore, has a lower quenching efficiency.
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.
To ensure effective capture on XanTec’s streptavidin-modified sensor chips, the ligand should be biotinylated at a substitution level of one biotin molecule per ligand or lower. Since commercial biotinylation protocols often result in higher degrees of labeling, it is recommended to use an NHS-biotin reagent at a concentration of 1.1 moles of biotin per mole of ligand or less.
It‘s crucial to thoroughly remove any unreacted biotinylation reagent from the ligand solution before capture to avoid competition with the biotinylated ligand for binding sites on the streptavidin chip. This can be achieved through size-exclusion chromatography, and for optimal results, the purification process should be repeated at least once to ensure complete removal of free biotin.
Failure to properly remove excess biotin can significantly reduce binding efficiency, so taking these extra steps will help ensure robust and reliable immobilization.
Allow the sealed sensor chip pouch to equilibrate at room temperature to prevent condensation on the chip surface.
After opening the pouch, install the sensor chip by following 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 running buffer like HBSTE or HBSTE+ and mount a compatible XanTec streptavidin sensor chip. | ||
| 2 | Condition all channels with Conditioning buffer 1. Wait until the baseline has stabilized. Note: Shorter or no conditioning can increase the immobilization yield but results in a less stable baseline. |
15 | 600 |
| 3 | Divert your reference channel and inject your biotinylated ligand at concentrations of 1–100 nM. Low ligand concentrations and flow rates are recommended to achieve good control over ligand immobilization levels. Adjust conditions and repeat ligand injection as required to achieve the target immobilization level. Wait until the baseline has stabilized. |
1–10 | 60–900 |
| 4 | Optional: Inject biotin blocking solution to quench remaining streptavidin binding sites. Note: In SPR instruments with serial flow cells, the biotin blocking solution may carry over to adjacent flow cells, potentially decreasing ligand immobilization capacity in these cells. | 15 | 300 |
| 5 | Start Interaction analysis. We recommend to begin with 3–5 consecutive regeneration cycles to improve data quality. |
Tween containing running buffers like HBSTE or HBSTE+ are generally recommended for streptavidin-modified sensor chips from XanTec.
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 like HBSTE.
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 oor 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 [1][2]. 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.. |
The storage and reuse of XanTec’s streptavidin sensor chips are not recommended, as the captured ligand gradually leaches from the chip surface, reducing immobilization capacity and potentially causing cross-contamination of the sensor surface‘s reference areas.
| Issue | Possible solution |
|---|---|
| Insufficient ligand immobilization level | Make sure that the XanTec streptavidin sensor chip provides a sufficient immobilization capacity. Otherwise, switch to a chip variant that provides higher immobilization levels. Make sure that all interfering components of your ligand stock solution (such as free biotin) are completely removed before immobilization. Sometimes, multiple desalting steps are necessary for quantitative removal. Increase the biotin ligand contact time. Increase the concentration of your biotinylated ligand. |
| Insufficient ligand activity | Check ligand and analyte integrity in your stock solutions and in the immobilization buffer with regard to activity, aggregation, and biological contamination. Reduce the molar excess of the biotinylation reagent used during the ligand biotinylation process. If applicable, switch to a site-directed ligand biotinylation approach. If the ligand was purchased pre-biotinylated, inquire about the degree of biotinylation. Many commercially available formulations have biotinylation levels that are unsuitable for SPR applications. Decrease the overall immobilization level to minimize ligand crowding. |
| Ligand leaching | If high ligand immobilization levels are used and your SPR system employs a serial flow cell configuration, the biotinylated ligand may gradually leach from the immobilized channel, leading to cross-contamination of the reference channel. In such cases, it is recommended to block the reference channel with water-soluble biotin analogs and reduce the ligand immobilization levels. |
V. 10/24a
For in-vitro use only. Not for use in clinical diagnostic procedures.