Sensor chip SA with pre-immobilized Streptavidin is a general-purpose sensor chip for capture of biotinylated ligands. A combination of high affinity (KD » 10-15) and binding capacity, reproducibility and chemical resistance gives excellent performance in a broad range of applications. Examples include the capturing of large amounts of PCR products to facilitate detection of small molecules, as well as the use of harsh regeneration protocols with retained cycle-to-cycle reproducibility.
The ligand concentration to be captured may be as low as pM (1). Short oligonucleotides and other ligands often bind rapidly to the Streptavidin and equilibrium is achieved in a short time. For longer nucleic acid fragments, low flow rates (2-5 μl/min) and longer contact times are used (up to 30 minutes) and 0.5 M NaCl is included in the sample buffer. To control immobilization levels use short contact times with diluted samples.
Instead of purchasing sensor chip SA, it is possible to make a sensor chip with Streptavidin from a sensor chip CM5 (2). After activation of the CM5 chip with NHS/EDC, inject 40 μl of 100 μg/ml Streptavidin in 10 mM NaAc, 0.1 mM EDTA, 1 mM NaCl, 1 mM DTT pH 4.6. The flow rate is 5 μl/min. After ethanolamine deactivation, the biotinylated protein of interest can be injected. Unbound proteins can be removed with a pulse of 2 M NaCl at 20 μl/min.
The biotin-(strept)avidin binding is resistant to a wide range of agents. Choice of the regeneration procedure may depend on the stability of the ligand coupled. For regeneration of short oligonucleotide ligands, 1 mM HCl is recommended. For longer oligonucleotides and proteins bound to nucleic acids, 1 M NaCl in 50 mM NaOH can be used.
Regeneration solutions may dissociate a little of the ligand from the chip. A short injection with biotinylated ligand will restore the original surface concentration.
|(1)||Nilsson, P. et al Real-time monitoring of DNA manipulations using biosensor technology. Analytical Biochemistry 224: 400-408; (1995).|
|(2)||O'Shannessy, D. J. et al Immobilization chemistries suitable for use in the BIAcore surface plasmon resonance detector. Analytical Biochemistry 205: 132-136; (1992).|