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BIACORE Sample Preparation

When planning a BIACORE experiment, it is important to carefully consider which of the molecular partners of interest should be used as the LIGAND (immobilized partner) and which should be used as the ANALYTE (fluid-phase partner).  Important considerations are:

1. Molecular weight and the ratio of the molecular weights of the ligand-analyte pair.

The BIACORE is a mass sensor.  Larger molecules give higher signals than smaller molecules and for the same immobilization level, smaller molecules represent more binding sites than larger molecules.  Hence, when possible, one should consider using the smaller molecular weight partner as the ligand, particularly when there is a large discrepancy between the molecular weights of the ligand and analyte being studied.

2. The ability to couple or capture the molecule.

In order to use a molecule as a LIGAND, one needs some way of immobilizing it to the sensor surface.  This may be done by covalently coupling the LIGAND to the sensor surface using amine-specific coupling, thiol-specific coupling, or some other coupling chemistry.  Naturally, this requires the presence of the proper reactive group in the potential LIGAND molecule.  Small molecules may or may not have the appropriate groups available for coupling.  Alternatively, one can use a specific, non-inhibitory capture molecule that can itself be coupled to the surface (i.e. an antibody) and used to subsequently capture the LIGAND.  This requires a very stable interaction between the capture molecule and the LIGAND.  A slow off-rate of the LIGAND is desirable to enable accurate kinetic studies of the LIGAND - ANALYTE interaction with minimal (controllable) contribution from the capture molecule - LIGAND interaction.

3. Purity.

Typically, for general binding studies, high purity (~90% homogeneity) of the LIGAND molecule is more important than high purity of the ANALYTE molecule.  However, there are exceptions where pure LIGAND may not be an absolute requirement (i.e. if one is using a "well-behaved" specific capture molecule to capture the LIGAND out of a crude mixture).  In contrast, kinetic studies require high purity of both the LIGAND and the ANALYTE.

4. Availability.

LIGAND utilization is generally less than ANALYTE utilization.  Thus, if one has a precious supply of one of the molecular partners, it may be more economic to use this molecule as the LIGAND (if possible based on the above considerations).  Once again, however, there are exceptions.  If one is using a capture molecule, then repeated cycles of stripping and re-capture of fresh LIGAND dramatically increases the utilization of LIGAND.  Kinetic studies generally use more material as well, due to the requirement for numerous cycles at various ANALYTE concentrations and LIGAND coupling densities to verify the measured kinetic parameters.

Once the LIGAND and ANALYTE have been decided upon, samples should be prepared with the following considerations in mind:

1. LIGAND (FOR COUPLING TO SURFACE).

The LIGAND buffer should not contain any primary amines (e.g. Tris, glycine, ethanolamine, carrier protein, etc.).  These will all interfere with the coupling reaction.

The salt concentration should be kept near physiological (~150 mM).

It is very useful to know the molecular weight of the LIGAND, as well as its pI to determine the capacity of the chip and the optimal coupling pH.

Good concentrations for coupling are typically between 0.1 and 1 mg/ml.  Only a small amount will likely be used (< 100 microliters).

2. ANALYTE.

Buffers should contain at least 150 mM salt, with little or no glycerol/carrier protein to minimize refractive index changes.  Low levels (0.005%) of non-ionic detergent (P20 or Tween20) should be added to help reduce non specific binding.

Precise knowledge of the ANALYTE concentration is required for kinetic studies, and knowing the molecular weight is useful.

The concentration of ANALYTE required varies considerably, especially for kinetic studies.  However, good starting concentrations are between 0.1 and 1 mg/ml.

All running buffers should be filtered through 0.2 micron filters, and degassed thoroughly.

Dr. Padmaja Mehta-D'Souza