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Instructions
for use

RG-modifier (PDF download)

Product description

Introduction

The RG-modifier is a 24-nt, single-stranded, azido-modified DNA oligomer designed for efficient ligand immobilization. It can be readily coupled to dibenzocyclooctyne (DBCO)-modified ligands, enabling stable (yet fully reversible) binding to RG-modified sensor chips such as RGD200M under physiological conditions. This makes it a highly versatile tool for ligand immobilization, particularly if assay development time is limited or if the use of the RG-SA reagent (streptavidin-DNA oligomer conjugate) is not feasible. This may be the case if:

• The ligand is not biotinylated and biotinylation is not an option.
• The low (but significant) drift that streptavidin-biotin coupling introduces complicates the evaluation of tight binders.
• Streptavidin nonspecifically interferes with the biological system.
• The immobilization capacity of RG-SA is insufficient for the intended application.

Ligand conjugation via the RG-modifier is achieved through a highly selective, one-step click reaction with a DBCO-functionalized ligand. Due to its exceptional specificity, this reaction eliminates the need for additional purification, thereby streamlining the workflow.

With its rapid, efficient, and reversible immobilization capabilities, the RG-modifier represents a powerful solution for demanding experimental setups. It offers researchers greater flexibility, time and cost savings plus enhanced reliability in ligand-capturing strategies.

Related documents

Instructions for Use – DBCO labelling kit
Instructions for Use – RG Sensor Chips

Additional materials required for RG conjugation

A ligand bearing DBCO-functionalities (to be provided by the user. Can be prepared using the DBCO labelling kit from XanTec).

Preparations for RG conjugation

Ligand DBCO-functionalization

If your ligand does not come with DBCO-functionality, it needs to be DBCO-functionalized prior to RG-modification. A requirement for successful DBCO conjugation is that the ligand contains at least one NHS-reactive amino group. In this case, the ligand can be modified with the DBCO-PEG₄-NHS ester as part of the DBCO labelling kit (product code K DCL-3C).

Accurate estimation of the molar excess of DBCO-PEG4-NHS ester relative to the ligand is crucial to prevent crosslinking of DBCO-labelled ligands on the sensor chip. For proteins, which typically have multiple reactive amines (e.g., N-terminus and lysine side-chains), a degree of labelling (DOL)¹ between 0.2 and 0.5 strikes a good balance between maximizing labelled protein and minimizing multivalent labelling. At protein concentrations ~50 µM, a sub-stoichiometric molar ratio of DBCO-PEG₄-NHS ester of 0.4–0.8 is recommended to achieve this range. At lower protein concentrations (e.g., 15 µM), increase the molar ratio of the DBCO-PEG₄-NHS ester to 0.8–1.0 to compensate for the increased proportion of hydrolyzed NHS ester. For further details, please refer to the Instructions for Use of the DBCO labelling kit.

Determine the DOL of your DBCO-conjugate via UV spectroscopy. Measure the absorbance (A) of the ligand solution at 280 nm and 309 nm. Use the following equation to calculate the degree of labelling:

R = A(309 nm)/A(280 nm)
εP,280 = Molar extinction coefficient of the unmodified ligand at 280 nm [M⁻¹ cm⁻¹]
εP,309 = Molar extinction coefficient of the unmodified ligand at 309 nm [M⁻¹ cm⁻¹]
εL,280 = Molar extinction coefficient of the DBCO-linker at 280 nm = 12000 M⁻¹ cm⁻¹
εL,309 = Molar extinction coefficient of the DBCO-linker at 309 nm = 12000 M⁻¹ cm⁻¹

¹ The degree of labelling (DOL) is the average number of label molecules (e.g., DBCO) attached to each target molecule.

Protocol for RG conjugation

V. 02/25a

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