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Instructions
for use

RG Sensor Chips (PDF download)

Product description

Product code Prefix (designates the instrument): SCB, SCBS, SCBN, SPP, SCBI, SPSM, SCH, SPMX, SCR, SCS, SD +
Add: RGD200M
Example: SCBS RGD200M
Intended purpose Reversible immobilization of biotinylated ligands by using the RG biotin Capture reagent.
Reversible immobilization of ligands that were previously modified with the RG-modifier.
Recommended applications include the investigation of biomolecular interactions involving
  • binding partners that are difficult to regenerate
  • unstable ligands that need to be replaced regularly
  • ligands that are difficult to immobilize via amine coupling
Not recommended for ligands with nucleic acid binding motifs.
Storage Store at -20 °C, desiccated over molecular sieve 4A
or at 2–8 °C in physiological buffer.
Related products
  • RG biotin Capture reagent, product code C RG-SA-50I
  • Conditioning buffer 1, product code B C1-50ML
  • RG-modifier, product code C RG-MOD-3NM
  • DBCO Labelling Kit, product code K DCL-3

Introduction

RGD200M sensor chips are coated with a bioinert carboxymethyldextran matrix, derivatized with a 24-nt single-stranded DNA oligo sequence. This oligo is capable of binding ligands conjugated to the complementary DNA sequence by hybridization of the two DNA strands. While the captured ligand is bound stably in physiological conditions, complete regeneration of the chip can be achieved by denaturation of the double-stranded DNA at alkaline pH, liberating the surface-bound oligo for another capture cycle. XanTec offers two variants for DNA-mediated, reversible ligand immobilization using the RGD200M sensor chip:

RG Sensor Chips
Variant 1: RG biotin capture reagent (streptavidin, preconjugated with the complementary DNA oligo) is captured on the RGD200M sensor chip by hybridization of the two single-stranded DNAs. This step is followed by the injection of a biotinylated ligand; such ligands bind to streptavidin with exceptional affinity and stability. The amount of immobilized ligand can be controlled by varying the injection time and ligand concentration. Once the baseline has stabilized, the surface is ready for biomolecular interaction experiments. Upon completion of experiments, the hybridized DNA oligo can be denatured by injection of Conditioning buffer 1 (1 M NaCl in 0.05 M NaOH) or – more stringent – Conditioning buffer 1 mixed with 30 % acetonitrile, which usually results in complete regeneration of the sensor chip for subsequent experiments.
RG Sensor Chips
Variant 2: In the first step, the ligand is conjugated with a DBCO (dibenzocyclooctyne) functionality (see Xantec DBCO Labelling Kit). After a quick desalting step, the DBCO-functionalized ligand is reacted with the Xantec RG-modifier DNA oligo by a highly selective click-reaction. The ligand can then be directly coupled to the RGD200M sensor chip via hybridization of the two DNA strands. The amount of immobilized ligand can be controlled by varying the injection time and ligand concentration. Once the baseline has stabilized, the surface is ready for biomolecular interaction experiments. Upon completion of the experiments, the hybridized DNA oligo can be denatured by injection of Conditioning buffer 1 (1 M NaCl in 0.05 M NaOH) or – more stringent – Conditioning buffer 1 mixed with 30 % acetonitrile, which usually results in complete regeneration of the sensor chip for subsequent experiments.

Compared to standard streptavidin-biotin coupling, RG Capture sensor chips used with the RG biotin Capture reagent offer a major advantage: significantly simplified ligand regeneration. By removing the entire streptavidin-biotin complex, these chips eliminate the need for time-consuming regeneration protocols, streamlining the experimental workflow.

Variant 2 goes a step further by removing streptavidin from the surface entirely. This results in even higher immobilization densities due to increased available volume within the sensor matrix, along with enhanced capture binding stability. As a result, these chips are particularly well-suited for interaction analyses involving smaller, tightly binding analytes.

Notes

RG sensor chips are not recommended for use with ligands or analytes known to nonspecifically interact with nucleic acids.

Reversible immobilization via RG biotin Capture reagent (variant 1)

Additional materials required

RG biotin Capture reagent (product code: C RG-SA-50I): 150 µL oligonucleotide-modified streptavidin conjugate (RG-SA, regenerable streptavidin) stock solution for reversible immobilization of biotinylated ligands on RGD sensor chips.

Biotinylated ligand (to be provided by the user).

Conditioning buffer 1 (product code: B C1-50ML): 0.05 M NaOH, 1 M NaCl.

Acetonitrile ≥99.5 % (optional, to be provided by the user)

Regeneration buffer: Conditioning buffer 1. Alternative: Conditioning buffer 1 mixed with acetonitrile (7:3 v:v). 1

1 Check instrument fluidics compatibility with acetonitrile in advance. The regeneration mix is stable for at least one week at room temperature.

Preparations for ligand capture

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.

Optional: Ligand biotinylation

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.

Preparation of Capture reagent working solution

Prepare the RG-SA working solution: Dilute the RG biotin Capture reagent stock solution 1:100 in physiological running buffer. This will produce a total of 15 mL RG-SA working solution (100 nM), which should be sufficient for at least 50 injections of 250 µL each. Typical streptavidin immobilization levels are 4000–5000 µRIU.

Optional: Preparation of Regeneration solution

Prepare the alternative regeneration solution by mixing 7 mL of Conditioning buffer 1 with 3 mL of acetonitrile. This regeneration solution is more stringent than Conditioning buffer 1. Prepare the solution fresh before use.

Sensor chip

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.

Protocol for ligand capture

Procedure Flowrate
[µL/min]		 Injection
time [s]
1 Equilibrate your SPR-system with physiological running buffer and mount an RGD sensor chip.
2 Condition the chip with Conditioning buffer 1.
Wait until the baseline has stabilized.		 25 5 × 60
3 Inject RG-SA working solution.
Wait until the baseline has stabilized.		 20 600
4 Inject 2–200 nM of your biotinylated ligand, preferably in running buffer. Immobilization levels can be controlled via the injection time and ligand concentration.
Wait until the baseline has stabilized 		10 120–900
5 Start your interaction experiments.
6 Regenerate the chip surface with Conditioning buffer 1, or the alternative regeneration solution containing acetonitrile. 25 2 × 60
7 Repeat steps 3–6 as required.

Notes

The coupling yield can be modified by changing the RG Capture reagent concentration, injection time, and/or flow-rate.

DNA-binding proteins cannot be analyzed using RGD sensor chips because of nonspecific interactions with the sensor chip coating.

Make sure that your buffers are freshly prepared and deoxyribonuclease-free.

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 deoxyribonuclease-free physiological buffer for storage.

Reversible immobilization via oligo-modified ligand (variant 2)

Additional materials required

RG-modifier (product code: C RG-MOD-3NM): 30 µL of 100 µM azide-modified complementary oligo sequence in 0.01 M HEPES and 0.1 M EDTA, pH 7.0.

DBCO-functionalized ligand (to be provided by the user, see DBCO Labelling Kit K DCL-3). Concentrations of ≥1 mg/mL are recommended.

Conditioning buffer 1 (product code: B C1-50ML): 0.05 M NaOH, 1 M NaCl.

Acetonitrile ≥99.5 % (to be provided by the user)

Regeneration buffer: Conditioning buffer 1. Alternative: Conditioning buffer 1 mixed with acetonitrile (7:3 v:v).

Preparations for ligand capture

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.

Optional: Ligand DBCO-functionalization

Ligand DBCO-functionalization is described in detail elsewhere (Instructions for Use – DBCO-labelling kit). In general, the procedure is similar to standard biotinylation via amine-reactive NHS ester.

The ligand should be DBCO-modified at a substitution level of one DBCO molecule per ligand or lower. Since commercial DBCO-functionalization protocols often result in higher degrees of labeling, it is recommended to use the DBCO-NHS reagent at a concentration of 1.1 moles of DBCO per mole of ligand or less.

It‘s crucial to thoroughly remove any unreacted DBCO reagent from the ligand solution before the DBCO-modified ligand reacts with the RG-modifier in a second step. This can be achieved through size-exclusion chromatography. For optimal results, the purification process should be repeated at least once to ensure complete removal of free DBCO.

Ligand preparation

Add RG-modifier stock solution to your DBCO-functionalized ligand solution and mix carefully. A molar ratio of 0.8:1 (RG-modifier:DBCO-labelled protein) ensures that no significant amount of free RG-modifier will be available after 16 h.

Note: Make sure that all solutions are free of sodium azide.

Optional: add 0.15 % ProClin 150 to avoid microbial growth.

Optional: remove free RG-modifier by using a small desalting column (cut-off >10 kDa).2

Dilute the oligo-modified ligand in running buffer as required (2–200 nM recommended). Storage conditions correspond to those of the unconjugated protein.

2 This step may be necessary in cases of incomplete click-reaction of the azide–oligo and the DBCO–ligand.

Optional: Preparation of Regeneration solution

Prepare the alternative regeneration solution by mixing 7 mL of Conditioning buffer 1 with 3 mL of acetonitrile. This regeneration solution is more stringent than Conditioning buffer 1. Prepare the solution fresh before use.

Sensor chip

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.

Protocol for ligand capture

Procedure Flowrate
[µL/min]		 Injection
time [s]
1 Equilibrate your SPR-system with physiological running buffer like HBSTE or HBSTE+ and mount an RGD sensor chip.
2 Condition the chip with Conditioning buffer 1.
Wait until the baseline has stabilized. 		25 5 × 60
3 Inject 2–200 nM of your oligo-modified ligand, preferably in running buffer. Immobilization levels can be controlled via the injection time and ligand concentration.
Wait until the baseline has stabilized. 		20 120–900
4 Start your interaction experiments.
5 Regenerate the chip surface with Conditioning buffer 1, or the alternative regeneration solution containing acetonitrile. 25 2 × 60
6 Repeat steps 3–6 as required.

Notes

The coupling yield can be modified by changing the oligo-modified ligand concentration, injection time, and/or flow-rate.

DNA-binding proteins cannot be analyzed using RGD sensor chips because of nonspecific interactions with the sensor chip coating.

Make sure that your buffers are freshly prepared and deoxyribonuclease-free.

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 deoxyribonuclease-free physiological buffer for storage.

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.

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.

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 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.
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 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 RG 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
Inefficient regeneration If the chip with bound streptavidin–ligand-complex or the oligo-modified ligand suffers from incomplete regeneration on injection of Conditioning buffer 1, injections of Conditioning buffer 1 premixed with 30 % acetonitrile (2 × 1 min) improves regeneration results.
Inefficient regeneration indicates over-biotinylation. Reduce the biotinylation level and retest regeneration efficiency.
High Nonspecific binding In some cases, nonspecific interactions between the analyte and the sensor coating can negatively affect the biomolecular interaction analysis. If the nonspecific interaction is caused by hydrogen bonds between free nucleic acid bases and the analyte, an increase of the RG-SA immobilization level can help to decrease the amount of nonspecific binding.
Add BSA to your running buffer with concentrations from 0.02–3 %.
Add herring sperm DNA fragments to the running buffer to compensate for nonspecific oligo binding.

V. 01/25a

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