| Product code | Prefix (designates the instrument): SCB, SCBS, SPP, SCBI, SPSM, SCH, + Add: LP or LD Example: SCBS LD |
|---|---|
| Intended purpose | LP: Reversible spreading of vesicles with or without membrane proteins under physiological conditions on a 2D carboxymethyldextran cushion containing lipophilic anchors. Vesicles form a supported lipid bilayer, preserving membrane protein activity. LD: Reversible capture of vesicles with and without membrane proteins under physiological conditions in a 3D carboxymethyldextran hydrogel, containing lipophilic anchors. The vesicle topology remains intact making LD sensor chips suitable for biomolecular interaction experiments involving transmembrane proteins like GPCR. |
| Storage | Store at -20 °C, desiccated over molecular sieve 4A or at 2 °C–8 °C in physiological buffer. |
| Related products |
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LP and LD sensor chips are coated with a bioinert 2D (CMDP) or 3D (CMD500L) carboxymethyldextran polymer matrix, partially functionalized with lipophilic alkyl anchor groups. In contrast to solely hydrophobic 2D chip surfaces, such as HPP, LP and LD sensor chips are partially hydrophilic and conserve the functionality of lipid bilayer structures.
On LP sensor chips, the lipophilic anchor groups on top of a hydrophilic CMDP cushion allow the reversible adsorption and rupture of lipid vesicles followed by formation of a supported lipid bilayer (SLB) [1]. Incorporated membrane proteins usually stay intact and do not lose their biological activity. These membrane proteins are available for biomolecular interaction analysis especially for moderate to high molecular weight analytes like proteins. LP sensor chips also are a convenient support for SLB model systems, providing the option of multiple regeneration.
LD sensor chips are coated with a hydrophilic, 3D hydrogel based on brush structured carboxymethyldextran. Lipid vesicles can diffuse inside the LD hydrogel matrix. Incorporation of the lipophilic anchor groups of the LD chip surface allows the reversible capture of such vesicles. The shape of the vesicles usually stays intact, making them suitable for the biomolecular investigation of membrane and transmembrane proteins like GPCR. In contrast to LP, the immobilization capacity of LD sensor chips is about 3–5 times higher, allowing for the investigation of biomolecular interactions including smaller analytes.
Freshly prepared solution of lipid vesicles (to be provided by the user): 0.5–1.0 mM of total lipid content is usually sufficient.
0.05 M Sodium hydroxide, (product code B NO-50ML): 50 mM NaOH
0.02 M CHAPS, (product code B CH 50ML): 20 mM CHAPS
Detergent-free running buffer like HBS, (10×HBS buffer, product code B HBS10-500ML)
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 which may otherwise disrupt the lipid bilayer or lipid vesicles. A subsequent blocking step with 1 mg/mL BSA helps to reduce nonspecific vesicle binding to the inner wall of the fluidic system.
Prepare a fresh lipid vesicle solution in a detergent-free physiological buffer, such as HBS. A total phospholipid-concentration of 0.5–1 mM is typically sufficient. The efficiency of adsorption depends on the vesicle size, with smaller vesicles being captured quicker than larger ones.
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 without detergent like HBS or PBS and mount a compatible XanTec LP or LD sensor chip. | ||
| 2 | Condition the chip surface by injecting a 20 mM CHAPS solution. Wash the fluidics thoroughly to remove CHAPS traces and allow the baseline to stabilize before proceeding. | 25 | 2×20 |
| 3 | Inject the freshly prepared vesicle solution at low flow rates. Capturing takes about 10–30 min to complete. Total signal increase should be 3000–6000 µRIU (RU) on LP sensor chips and up to 12.000 RU on LD sensor chips. LP: A signal decrease at the end of vesicle injection suggests vesicle rupture and the formation of a supported lipid bilayer (SLB). Spreading of the vesicles may take some time to complete and is indicated by a stabilization of the SPR-signal. LD: The lipid vesicle capture process is considered complete when the SPR signal transitions from a rising phase to a plateau, stabilizing at a constant response. |
2–10 | 600–1800 |
| 4 | LD only: If compatible with the lipid vesicle preparation, stabilize the baseline by washing the surface with a short pulse of 10–50 mM NaOH before initiating interaction analysis. | 25 | 20 |
| 5 | Start Interaction analysis. Make sure that your detergent-free running buffer is properly degassed and filtered, as microscopic air-bubbles have a strong tendency to stick to the hydrophobic anchor groups. If you encounter a high level on nonspecific binding, try to block free hydrophobic sites by adding 200 µg/mL BSA to your running buffer before starting analysis. |
For instruments recently used with samples known to be challenging to clean from the flow system, it is recommended to run multiple instrument cleaning cycles before proceeding with the protocol described above.
Avoid switching between experiments that use detergent-containing buffers and those involving lipophilic sensor chips, as even trace amounts of detergent can disrupt the immobilization process.
Small liposomes, such as those produced via the extrusion technique, tend to adsorb more rapidly to the lipophilic surface compared to larger aggregates.
Liposomes and vesicles can adhere non-specifically to the inner walls of the device fluidics, which can lead to clogging over time. It is therefore strongly recommended to clean the system thoroughly after each experiment with liposomes or lipid vesicles.
Surface-bound vesicles can be regenerated by selectively dissociating the bound analyte under carefully controlled conditions to ensure complete removal without compromising the vesicle‘s binding integrity. Given the diversity of vesicle compositions, regeneration protocols should be optimized empirically. A practical starting point includes adjusting the pH using 10–100 mM HCl or 10–100 mM NaOH or introducing up to 10 % ethanol or DMSO to the regeneration solution.
The vesicles (LD) or the SLB (LP) may be removed quantitively by a 3 min injection of 20 mM CHAPS followed by a 1 min pulse of 50 mM NaOH.
For future reuse, sensor chips can be stored either dry or in a wet state under physiological conditions. Carefully clean the sensor chip with 20 mM CHAPS and 50 mM NaOH to remove all traces of previous modifications resulting from vesicle or liposome capture. When handling the sensor chip, avoid touching the top coating with gloves or tweezers to prevent contamination or damage.
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 | Remove 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 or centrifuge liquid off. |
| 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 LP/LD sensor chip 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 | Remove 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, detergent containing 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 LP and LD 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 detergent traces, and carefully dry it using a jet of filtered air or nitrogen or centrifuge liquid off. Then, insert the chip according to the instrument manufacturer‘s instructions. |
| Issue | Possible solution |
|---|---|
| High non-specific binding | Try to block free hydrophobic binding sites on the LP or LD sensor chip with an initial injection of 0.2–1 mg/mL BSA or another, non-interacting protein. |
V. 01/25a
For in-vitro use only. Not for use in clinical diagnostic procedures.