| Product code | K TK-50I |
|---|---|
| Intended purpose | The Thiol Coupling Kit is designed to facilitate surface and ligand thiol immobilization on XanTec HC and CMD sensor chips. Active disulfide groups are introduced on the chip surface, which react covalently with thiol-containing ligands to form a stable disulfide bond. |
| Kit contents | Contains buffers and chemicals for 40–60 activations
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| Storage |
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| Related products |
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The Thiol coupling kit K TK-50I is designed to facilitate surface and ligand thiol immobilization on XanTec HC and CMD sensor chips. This aim is achieved by introducing active disulfide groups on the chip surface, which react covalently with thiol-containing ligands to form a stable disulfide bond. Thus, thiol coupling via disulfide groups offers an interesting alternative to classical EDC/NHS coupling.
The kit functions as an extension of the Amine coupling kit (AN). Hence, the Amine coupling kit AN or its components are required in conjunction with the Thiol coupling kit.
Sufficient electrostatic preconcentration is a prerequisite for protein immobilization, so preconcentration scouting of the ligand (see protocol below) should be conducted before immobilization. Small molecules can diffuse freely into the chip coating and do not require electrostatic preconcentration for immobilization.
Amine coupling kit AN, NHS (product code K AN-50I) contains:
Solid EDC·HCl (product code C EDCHCL-1G): 1 g of solid EDC·HCl (purity >99%)
Activation buffer (product code B MN-50ML): 0.05 M MES, 0.1 M NHS, pH 5.0, 50 mL
Borate elution buffer (product code B BELU-50ML): 0.1 M sodium borate, 1 M NaCl pH 9.0, 50 mL
Ethanolamine quenching buffer (product code B EA85-50ML): 1 M ethanolamine·HCl, pH 8.5, 50 mL
Polycarboxylate hydrogel sensor chip, such as CMD or HC
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 accessible thiol- or disulfide groups (to be provided by the user)
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.
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.
Use concentrated ligand stock solutions if available (≥1 mg/mL). Thereby, dilution of the ligand into coupling buffer is less affected by the pH and ionic strength of the stock solution.
If your protein (ligand) has a free thiol group, dilute your protein in a suitable coupling buffer to a final concentration of 10–100 µg/mL and omit steps 1–3. If your ligand does not have a free thiol group, follow the steps below from the beginning. Add Na2EDTA to a final concentration of 1 mM to your coupling buffer to prevent metal-induced oxidation of thiol groups.
| Procedure: Preparations for thiol coupling | |
|---|---|
| 1 | Dissolve DTT in water to generate a 1 M stock solution. It is recommended to make this solution fresh and to work under a fume hood. |
| 2 | Add DTT stock solution to your physiologically buffered (pH 7–8) protein solution (≥1 mg/mL is recommended) to a final concentration of 1–10 mM DTT. Incubate for 30 min at room temperature or on ice. |
| 3 | Use a gel-filtration column (e.g., Zeba Spin™) to remove free DTT, and dilute your ligand with thiol functionality into the coupling buffer. Usually, a concentration of 10–100 µg/mL in the coupling buffer results in sufficiently high coupling yields. To prevent sulfhydryl oxidation and the formation of disulfide crosslinks, the modified protein should be employed immediately in a conjugation reaction. |
| 4 | Dissolve 150 mg PDEA in 5 mL ultrapure water to produce a 160 mM solution. Prepare aliquots, and store the remaining material at -25 °C until use. Use within 3 months. |
| 5 | Allow the EDC to reach room temperature. Weigh out sufficient EDC to make an activation solution of 50 mM (HC) or 100 mM (CMD, CMDP). Use the weighed EDC within 30 min and store it in a dry place. Store the remaining EDC under inert gas and dry over molecular sieve 4A at −25 °C. Add EDC to the Activation buffer directly before use. |
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 not known, then consider undertaking 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 pH 4.0 is reached.
| 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. | ||
| 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 the 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 preconcentration effect. |
10 | 300 |
| 4 | Inject the Borate elution buffer. Wait until the baseline has stabilized. |
25 | 60 |
| Procedure for thiol coupling | Flowrate [µL/min] |
Injection time [s] |
|
|---|---|---|---|
| 1 | Mount a compatible XanTec sensor chip. Equilibrate your SPR-system with water as running buffer. | ||
| 2 | Condition the surface with Borate elution buffer. Wait until baseline has stabilized. |
25 | 3 × 60 |
| 3 | Mix solid EDC and Activation buffer to a final EDC concentration of 50 mM (HC) or 100 mM (CMD, CMDP). Filter the EDC/Activation buffer (0.45 µm) and inject. Wash briefly with water. | 15 | 600 |
| 4 | In the meantime, mix 160 mM PDEA solution with the Borate coupling buffer 1:1 to produce an 80-mM PDEA working solution. Inject the PDEA working solution to introduce reactive disulfide groups. | 15 | 300 |
| 5 | Wash briefly with water and inject the protein solution in suitable Coupling buffer. Protein concentrations of 10–100 µg/mL are recommended. | 15 | 600 |
| 6 | Quench the remaining reactive disulfide groups by injecting Capping buffer. | 15 | 300 |
| 7 | Optional: remove loosely physisorbed protein with Borate elution buffer. | 25 | 3 × 60 |
| 8 | Switch to physiological running buffer and wait until the SPR-signal has stabilized. |
Physiological running buffer can also be used instead of water during immobilization. However, the dispersion effects of the protein coupling solution and running buffer can lower immobilization yields somewhat.
Ensure that your physiological running buffer is free from reducing agents such as DTT or TCEP.
Higher immobilization yields can be achieved by increasing protein concentration and/or protein-contact time.
For later reuse, sensor chips can be stored dry or wet under physiological conditions free from reducing agents. When handling the sensor chip, avoid touching the top coating with gloves or tweezers. Storage of CMDP sensor chips is not recommended.
Biacore users only: Prevention of detachment of the glass chip in the instrument after chips have been stored under buffer or at 100% humidity is important. 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 can 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 ~60 min 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 stream 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 is dependent upon 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 instructions set by the instrument manufacturer. Protective top coating applied: Equilibrate the sensor chip to room temperature before opening the storage container. Immerse the chip in physiological buffer for 10 min to remove the protective layer from the hydrogel coating. Rinse gently with ultrapure water and dry it carefully 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 ultrapure 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 is dependent mainly on the stability of the immobilized ligand. The underlying hydrogel coating should be stable for several days-to-weeks under such conditions. Long-term storage in water is not advised because 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 ultrapure water to remove buffer salts, and dry it carefully using a stream of filtered air or nitrogen. Then, insert the chip according to instructions set by the instrument manufacturer. |
| Issue | Possible solution |
|---|---|
| Insufficient electrostatic preconcentration | Carry out electrostatic preconcentration screening to check for optional preconcentration conditions. Desalt protein against water or Coupling buffer to remove possible salt contaminants. Lower the ionic strength of the Coupling buffer. The ligand is too acidic (pI <3.0) and does not preconcentrate on polycarboxylate sensor chips. In this case, alternative coupling methods should be considered. If you observe an unusually high increase in the signal after EDC/NHS-activation, impure EDC might be the reason. Replace the EDC by a fresh vial. Decrease the EDC concentration. |
| Insufficient level of protein immobilization | Ensure that optimal conditions for electrostatic preconcentration have been chosen. Ensure that your ligand has a free thiol group that is available for coupling. Increase the EDC concentration. Increase the protein-contact time. Increase the protein concentration. |
| Insufficient ligand activity | Check ligand integrity in your stock solution with regard to activity, aggregation, and biological contamination. Reduce the EDC concentration to avoid ligand crosslinking. Reduce the overall immobilization level. Increase the pH of your coupling buffer if your protein is sensitive to acidic pH. Sometimes, the ligand couples at its binding site. In this case, alternative coupling methods should be considered. |
V. 01/25a
For in-vitro use only. Not for use in clinical diagnostic procedures.