I attempted to immobilize a biotinylated nucleic acid polymer on SA sensor chip using a default immobilization method (Biacore). The immobilization resulted in negative (!!) immobilization levels. What is the possible reason for negative immobilization levels? Does it mean that Streptavidin is leeching off the sensor chip surface? And how can I troubleshoot it?
Immobilizaton Parameters:
Chip: SA
Ligand: biotinylated nucleic acid polymer (commercially produced)
ligand conc = 10 nM, ligand MW = 5600 Da
Method: Wizard method for SA chip immobilization with default settings
Instrument: Biacore T200
Thank you for your help in advance.
One possibility is that your system is not very clean. Try the recommendations in page 3 of the instruction (see link below) before capturing.
https://www.gelifesciences.com/gehcls_images/GELS/Related%20Content/Files/1384878377977/litdoc22060734_20140515223917.pdf
Streptavidin is covalently attached to the sensor chip so couldn't have been removed under your condition.
To troubleshoot, inject some streptavidin onto the surface to see if you actually already have some ligand being captured even though you have a negative immobilization levels.
Did you do the pre-treatment of the streptavidin surface before capturing the biotinylated ligand? With this surface, it is critical to inject 50 mM NaOH before injecting the biotinylated ligand. Usually about five 1 min injections of 50 mM NaOH are needed to stabilize the surface before capturing the biotinylated ligand especially if the goal is to capture a very small amount of ligand.
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