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LABORATORY GUIDELINE 28-9615-84 Ver AB

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How to use Sensor Chip SA in BiacoreTM 3000 and BiacoreTM 2000 The Sensor chip SA surface is pre-immobilized with streptavidin and ready for high-affinity capture

of biotinylated ligands.

Surface preparation 1. Desorb the instrument prior to immobilization.

2. Create or download (www.Biacore.com/methods) a method using Method Definition Language

(MDL) that includes:

- Surface conditioning with 3 consecutive injections of 1 M NaCl in 50 mM NaOH. Flow rate

10 μl/min and contact times around 1 min is recommended.

- Immobilization of the biotinylated ligand. Concentrations may be as low as in the pM

range. Flow rate 10 μl/min or higher and contact times around 1 min is recommended.

- An extra wash with a solution of 50 % isopropanol, 50 mM NaOH and 1 M NaCl after

ligand injection. Use the command WASHPOS n/s (needle and sample loop will be

washed) followed by WASH n/s for subsequent wash with running buffer.

Note: For convenience, you may prepare a solution of 100 mM NaOH, 2M NaCl and mix 1:1 with

isopropanol when the wash solution is needed. The solution can be stored at 20° C for at

least one month.

Interaction analysis

1. Inject the analyte. Flow rate and contact time will depend on application requirements (e.g.

Kinetic analysis: Flow rate 30 μl/min and contact time ~2min).

2. Regeneration of analyte from ligand. Generally, use same flow rate as for analyte injection and

contact time 30-60 s.

- For detailed information on regeneration strategies refer to Sensor Surface Handbook

(BR-1005-71).

How to use Sensor Chip SA in Biacore 3000 and Biacore 2000 Ver AB

Important considerations

• Use running buffer containing detergent (e.g. HBS-EP+) unless the ligand or analyte is detergent

sensitive. If you use PCR products as analytes, include ≥ 0.5 M NaCl in sample buffer and use

longer contact times, up to 30 min.

• For applications that use a reference surface, it is normal to use an unmodified Sensor Chip SA

surface. If available though, inject a biotinylated dummy protein (a protein that do not bind to

the analyte) followed by buffer over the reference surface.

• Always try to avoid blocking with free biotin:

- If blocking is needed, a recommended blocking agent is amino-PEO-biotin at 50 μM in

running buffer, since unmodified biotin is not sufficiently soluble in aqueous buffers to

provide satisfactory blocking conditions. Inject over both active and reference surface.

Flow rate 30 μl/min and contact time 1 min can be used as starting values. Always add

an extra wash with a solution of 50 % isopropanol, 50 mM NaOH and 1 M NaCl after the

blocking injection. Use the command WASH POS n/s (needle and sample loop will be

washed).

Note: Be aware that the biotinylated blocking agent may cause carry-over. The blocking agent

is normally a smaller molecule (more hydrophobic) than the biotinylated ligand. Small

molecules have an increased tendency of non-specific adsorption.

For contact information for your local office, please visit www.gelifesciences.com/contact

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