SPR Technology FAQ - MUSC ProteoGenomics Facility

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Useful information pertaining to SPR experimentation:

I. BUFFERS

  • Primary amine buffers must be avoided for amine coupling. It is important that there are no competing amine groups in the buffer (i.e. Tris or glycine, BSA, etc). For example,Tris and TBS buffers cannot be used due to the excess binding of free amine groups in the immobilization procedure.
  • HEPES is strongly recommended as a buffer at 10mM HEPES, pH 7.4 / 150mM NaCl.
  • Buffers containing glycerol or sucrose cannot be used. These components may alter data due to their elevated refractive indices.
  • The salt content of buffer should be < 200 mM.

    • » Running Buffers

  • An NaCl concentration of 150 mM or greater is helpful in reducing charge effects.
  • All buffers should be filtered with 0.2 m M filter and thoroughly degassed.
  • Low levels of nonionic detergent (i.e. 0.005% P-20 or Tween-20) should be incorporated.

II. ANALYTE – the molecule to be passed across a chip immobilized ligand.

  • Purity may be evaluated by SDS-PAGE and Coomassie staining.
  • The BIAcore staff may request such verification.
  • For kinetic studies, purity should be > 90%
  • It is important that the molar concentration of the analyte is known. This will ensure an accurate kinetic analysis.
  • For kinetic studies, the molecular weight of the analyte should be 2kD or greater.
  • The concentration should be 0.1 -1 mg/ml, if possible (total consumption depends on affinity, typically 10-100ug).
  • If glycerol is present in samples it should be less than 5%.
  • The exact concentration and molecular weight are desirable in interpreting results.
  • Proteins with pIs greater than 9.0 show a high degree of non specific binding to carboxydextran surfaces.

III. LIGAND – the molecule to be coupled to the surface of the sensor chip.

  • Most proteins are readily coupled though free amines (lysines or amino-terminus). There are, however, other coupling chemistries available, including thiol and aldehyde. There are also capturing schemes that employ biotin, GST, 6xHis, or antibodies. 
  • Ligand concentrations should be between 0.5 - 5 mg/ml (sample consumption ~10ug total).
  • It is also necessary to know the approximate molecular weight and pI of the molecule.

IV. MISCELLANEOUS

  • General BIA information can be found on the BIAcore web site