Preconcentration

In a pre-concentration experiment, the ligand is diluted in several immobilization buffers that differ by a half or one pH unit. The optimal buffer for the immobilization will be the one that gives a good pre-concentration but has the highest pH. In the figure ‘Pre-concentration’, pH 4.5 gives a fast and high pre-concentration. In some cases, however, pre-concentration is efficient but the covalent linking is not, due to the low pH. In these cases, the pH of the immobilisation buffer should be increased (in the example above to pH 5.0). If immobilization is still impossible, consider another immobilization method.

The concentration of the ligand also determines the pre-concentration rate. In general a concentration between 5 and 25 µg/ml of protein should be sufficient. A low pre-concentration and binding condition can provide more control over the amount of immobilized ligand. High pre-concentration values do not guarantee that the ligand will bind in large amounts. Other factors such as activation and availability of binding sites on the ligand will influence immobilization efficiency. In addition, rapid pre-concentration rates can create surfaces that have high drift or low biological activity after immobilization.

Dilution of ligand in the coupling buffer may have some undesired effects when the ligand is not concentrated enough. The ligand buffer will have an effect on the coupling buffer by altering the salt concentration and the pH. Use highly concentrated ligand stock solutions to dilute in the coupling buffer or use alternative methods to transfer the ligand in the coupling buffer, such as gel filtration or dialysis. Generally when the pI of a protein is known, the immobilization buffers are chosen 0.5 pH units below the pI for pI 3.5-5.5, 1 pH unit below pI for pI 5.5 - 7 and pH 6 for a pH over 7.

References