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Notes in immobilization and screening experiment

In order to reduce the background (reduce non-specific adsorption), the following cautions are required in the experiments.

Immobilization

  1. Perform centrifugal separation.

    The nano size of FG beads® gives them excellent dispersibility. As a result, magnetic separation in an organic solvent may be difficult, and it is necessary to recover the FG beads® by centrifugal separation.

  2. Disperse the beads well.     

    Centrifugal separation causes the FG beads® to agglutinate strongly, making it difficult to disperse them. Ordinarily a manual dispersion method or ultrasound would be used to disperse the beads. Although ultrasound separates the beads easily, caution is required due to the possibility of damaging the proteins. Therefore in general it is recommended that ultrasound be used when binding low molecular weight compounds, and that manual dispersion be used when binding proteins. The manual method involves resting the bottom of the micro tube in a plastic test tube stand and moving it roughly to disperse the beads. Depending on the type of micro tube, when using the manual method to disperse the beads, the bottom of the tube may crack or leakage from the lid may occur. Use a micro tube that is strong and has a lid with a tight fit. We recommend the use of cap locks.

     

Screening

  1. Perform magnetic separation

    When centrifugal separation is performed, heavy proteins and insoluble proteins are also precipitated, raising the level of the background. Because FG beads® have high dispersibility, magnetic separation requires time (in some cases 5 minutes or longer). However using magnetic separation avoids the risk of intrusion by these impurities and provides clear results with a low background level.

  2. Disperse the beads well

    If dispersion is insufficient following the FG beads® washing process after the binding reaction with the proteins, there is the possibility of impurities remaining inside the bead clusters. Therefore it is necessary to disperse the beads well. Use the manual method to disperse the beads. (With ultrasound, there is the risk that the proteins will be damaged.)