4 - Precondition and Preconcentration

4.1 Precondition

  • Prepare your sensor surface after docking a new sensor chip.
  • The precondition methods will prevent air-bubbles disturbing your injections during surface creation and further experiments.
  • Additionally the sensor surface is equilibrated.

Table 4-1: Overview of Pre-condition methods and the corresponding type of sensor

Method Details
Pre-condition for Amine/ HCA -Sensor The method contains repetitive cycles of elution buffer (1 M NaCl, 10 mM NaOH) and 100 mM HCl.
Both solutions will be addressed on all spots followed by all individual spots.
Pre-condition for BTC-Sensor The method contains repetitive cycles of elution buffer (1 M NaCl, 10 mM NaOH) and running buffer.
Both solutions will be addressed on all spots followed by all individual spots.
Pre-condition for HTC-Sensor The method contains repetitive cycles of 50 mM EDTA and running buffer.
Both solutions will be addressed on all spots followed by all individual spots.
Pre-condition for Pro-AG-Sensor The method contains repetitive cycles of regeneration (e.g. 10 mM Glycine, pH 1.5) and running buffer.
Both solutions will be addressed on all spots followed by all individual spots.

4.2 Preconcentration

The preconcentration procedure can be used to find the best immobilization buffer/pH while using carboxyl group-based sensor chips.

Procedure:

  • Dilute your target protein in a low ionic strength buffer with a pH, lower than the pI (isoelectric point) of your target protein and inject it over the non-activated sensor surface.
  • Due to charge-based attraction between surface sample (pos. charged) and sensor surface (neg. charged) the signal will differ depending on the pH of the used pre-concentration buffer.
Figure 4-1: Screenshot of the patent 5,716,854 describing the workflow of the preconcentration of a protein to a sensor surface

Rule of thumb: The optimal pH is 0.5-1 pH units below the pI. Note: Lower pH may partially or fully denaturate your protein. Thus may include according controls to check for retained protein activity, once it is attached to sensor surface.

Figure 4-2: Typical sensorgram of a preconcentration. Acetate buffer pH 5 should be taken as it shows similar signal height and slope as for pH 4.5, but pH 5 will be less harsh (acidic) for the protein itself

Note:

  1. at pH > 3.0 dextran matrix is negatively charged
  2. Immob.-buffer pH:
    • lower than pI of protein
    • higher than 3.0
Continue to 5 - Immobilization