Pre-concentration is the procedure used to obtain a high local protein concentration at the sensor chip surface. Pre-concentration makes the immobilization of the protein to the sensor chip surface more efficient. Pre-concentration is done with the dextran-carboxyl group based sensor chips. For sensor chips NTA, SA and hydrophobic based sensor chips pre-concentration is not beneficial because the ligand binding is based on pre-immobilized tags. Pre-concentration is aided by a low ionic strength and a solution pH slightly lower than the pI of the ligand (1). The pH of the coupling buffer, which will change the surface and ligand charges, regulates the rate and amount of the ligand that goes towards the sensor chip surface (pre-concentration). If the buffer has a high ionic strength, it will mask the charges of the surface and ligand and thereby diminish the pre-concentration effect. Therefore a buffer with low ionic strength (e.g. 10 mM) is used.
In general, an immobilization solution with a pH between 4.0 and 6.0 is used. The dextran matrix will lose its pre-concentration capacity at a pH lower than 3.0. The buffer components of the flow and immobilization buffer should not react with the activated surface. In addition, additives such as azide must be avoided during the immobilization because they can react with the activated surface sites and therefore lower the immobilization efficiency.
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.
|Flowbuffer||10 mM HEPES, 150 mM NaCl, 3.4 mM EDTA, 0.005% P20, pH 7.4|
|Malate buffer (0.5M)||Maleic acid; Mr 116
Dissolve 2.9 gram in 50 ml H2O; use 10 mM
|Acetate buffer (0.5 M)||NaAcetate; Mr 82.03
Dissolve 2.05 gram in 50 ml H2O; use 10 mM
|Formate buffer (0.5 M||NaFormate; Mr 68.01
Dissolve 1.7 gram in 50 ml H2O; use 10 mM
|EDC||N-ethyl-N’-(dimethylaminopropyl)carbodiimide); Mr 191.7
Dissolve 750 mg in 10.0 ml H2O to obtain 0.4 M.
Make aliquots of 200 µl and cap. Store at -20°C up to 1 year.
|NHS||N-Hydroxysuccinimide; Mr 115.09 Dissolve 115 mg in 10.0 ml H2O to obtain 0.1 M. Make aliquots of 200 µl and cap. Store at -20°C up to 1 year.|
|Ethanolamine||ethanolamine hydrochloride; Mr 61.08 Dissolve 611 mg in 10.0 ml H2O to obtain 1 M. Adjust pH to 8.5 Make aliquots of 200 µl and cap. Store at -20°C up to 1 year.|
|(1)||BIACORE AB BIACORE Getting Started. (1998).|