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Principles of surface regeneration

Choice of regeneration solution

Below are some recommendations for regeneration solutions based on experience at Biacore. For molecule types that are not specifically mentioned here, an empirical approach is the best route to success.

Always make sure that the regeneration solution is fully compatible with the running buffer, so that precipitation does not occur at interfaces between the two solutions. For example, MgCl₂ can cause precipitation with phosphate buffers, and SDS may precipitate if the potassium content of the running buffer is too high. Use a different running buffer if precipitation occurs on mixing with the regeneration solution.

Protein surfaces

Antibody ligands can usually be regenerated using acidic conditions (10 mM glycine-HCl, pH 3.0–1.5).

Although the range of protein-protein interactions studied with Biacore is very broad, experience has shown that a relatively small set of conditions provides for regeneration. Many surfaces are regenerated satisfactorily by one of the following solutions:

• 10 mM glycine-HCl pH 3.0–1.5 (available as ready-to-use solutions from Biacore
• 50–100% ethylene glycol
• 1–100 mM NaOH
• 1–4 M MgCl₂
• 0.5–5 M NaCl
• 0.02–0.5% SDS

Nucleic acids

Hybridized oligonucleotides can usually be regenerated with freshly prepared 1 mM HCl.

Proteins can be removed from RNA or DNA surfaces with 50 mM NaOH containing 1 M NaCl, or with 0.2–0.5% SDS.

Small molecules

Small molecules are generally more tolerant of harsh conditions than macromolecules, so that a wider range of regeneration conditions can often be considered. Solutions that have proved successful in regenerating low molecular weight ligands with protein analytes are:

• 20–100 mM NaOH in 30% acetonitrile. This solution is not stable and must be prepared fresh each day.
• 20–100 mM NaOH containing 0.5% Surfactant P20 or 0.05% SDS
• 10 mM glycine-HCl pH 3.0–1.5 (available as ready-to-use solutions from Biacore
• 1–4 M MgCl₂

Membrane-associated ligands

Regeneration of the Sensor Chip HPA surface is directed towards removing bound analyte from the ligand, and uses the same approach as for ligands covalently attached to CM-series and other sensor chips (with the exception that regeneration solutions should not contain detergents or organic solvents). Lipid monolayers composed of synthetic lipids such as DMPC and POPC are generally quite robust in this respect and can withstand exposure to e.g. 100 mM HCl and 100 mM NaOH.

Although the lipid monolayer with associated ligand and bound analyte can in principle be removed from Sensor Chip HPA by washing with detergents or organic solvents, this approach to regenerating the hydrophobic surface is not recommended and Biacore does not guarantee the performance of Sensor Chip HPA treated in this way.

Proteoliposomes and membrane vesicles are often difficult to remove completely from Sensor Chip L1, and regeneration of this kind of surface should be directed towards removing bound analyte from the ligand (using the same approach as for ligands covalently attached to CM-series and other sensor chips, with the exception that regeneration solutions should not contain detergents or organic solvents).

For surfaces prepared with pure liposomes, a 15–60 second injection of 40 mM octylglucoside is usually sufficient to strip the lipids from the surface. Other regeneration solutions that may be useful are 2:3 isopropanol:50 mM NaOH or 40 mM octyleneglycol:20 mM CHAPS. Removal of the lipid is generally recommended for applications that address interaction of the analyte with the lipid bilayer itself, such as studies of absorption of small molecules into membranes.

An OSR surface is regenerated with the aim of removing analyte and other bound sample components while leaving the reconstituted membrane intact. Detergents and organic solvents that may destabilize the membrane should be avoided.

Special cases

For some studies, the nature of the interaction points the way to a suitable regeneration approach which is specific for that interaction. Interactions that require metal ions, for example, can often be regenerated with chelating agents such as EDTA or EGTA, provided that the ligand survives removal of the metal ion. A specific example of this approach is Sensor Chip NTA, which uses Ni²+ ions chelated by nitrilotriacetic acid to capture his-tagged ligands, and which can be regenerated with EDTA that strips the Ni²+ ions from the surface.

Other regeneration strategies

Some ligand-analyte interactions may be difficult to regenerate with the conditions suggested above. Other conditions that have been found useful include:

• 10–100 mM HCl
• ~0.1 % trifluoroacetic acid
• ~1 M formic acid
• ~1 M ethanolamine-HCl at pH 9 or higher

In particularly stubborn cases, or when several components from the sample may bind to the surface, regeneration cocktails using mixtures of different types of solution (e.g. acidic and chaotropic conditions) may prove fruitful. The control software for most Biacore systems supports regeneration with two consecutive injections, and this approach can in some cases be an alternative to mixed reagents.

Further tips

Use your knowledge and experience of the ligand and analyte to guide your initial choice of regeneration conditions. For molecules used in affinity chromatography contexts, conditions for elution from the chromatographic medium can provide a starting point for scouting for regeneration conditions.

See also

List of help files

General recommendations for regeneration

Determine suitable regeneration conditions

Assay setup for regeneration scouting