i have been trying to immobilise an antibody on CM5 chip using Specify contact time Using the same amount of Antibody sometimes I get 5000RU immobilization, sometimes 10,000 RU... I am not sure why. Any inputs ... I use Biacore T200 instrument the method remains the same in all certainty!
Can you check the pre-concentration rate of the different immobilizations? You can set a report point and read the slope in RU/s. I gues they will be different.
One other question: Are you immobilizing the same antibody and the same batch? I can imagine that there will be differences in immobilization efficiency between antibodies and possible batch to batch differences (stock concentration, buffer composition).
I would use the option to immobilize a target amount of ligand. In our hands it is fairly consistent. And because the procedure checks the pre-concentration it will compensate for a slow or fast immobilization.
hey I tried that as well, I aimed for 8000 RU rather than Specify contact time. It is a very unseen problem (for me). The pre concentration says it is successful but the Immobilisation to reach the target fails. It either says response decrease or out of ligand!
The immobilization uses the pre-concentration phenomenon to provide for a high ligand concentration at the sensor surface. However, this does not mean that the ligand is covalently bound at the same level.
1), the composition of the original ligand solution is important. It should not contain any reactive components. In the case of an amine coupling this means no free amines such as found in TRIS or Azide. Although the pre-concentration can be high these compounds compete for the reactive sites on the sensor chip.
2), the ligand should have reactive sites. In the case of the amine coupling these are mainly the free amines on the exposed lysine residues. If the reactive amines are in or close by the analyte binding site, the interaction between the ligand and analyte will be lower than expected.
3), the activation of the surface should be reproducible. Use fresh prepared NHS and EDC (you can prepare and store aliquots at -20°C for up to one year) and monitor the activation response. It should be between 100 – 130 RU for a fresh CM5 sensor chip after 7 minutes of activation. The activated groups are labile and deteriorate rather quickly, so the ligand injections should be started immediately after activation. In addition, only activate one channel at the time.
4), the nature of the ligand, the pH of the immobilization solution and the sensor surface are of great influence on the pre-concentration and the possible covalent reaction. Before attempting an immobilization, check the pre-concentration of the ligand at different pH. In general a pre-concentration slope of 25 – 50 RU/s is sufficient to immobilize up to 5000 RU of ligand within 50 – 70 µl injection volume. Although higher pre-concentration rates can give higher immobilization levels, they sometimes suffer from crowding problems leading to a lower level of covalent immobilized ligand.
Checking the pre-concentration can also reveal strange behaviour of the ligand. Some ligands (e.g. with pI > 8.0) are known to stick to the dextran matrix. At the end of the pre-concentration they will not wash away and even an injection with 50 mM NaOH or 0.1 M HCl will not regenerate the surface.
We also encountered immobilizations that failed. The exact cause is not always clear but has probably to do with the immobilization algorithm in the software. When the last injection gives a lower response, the routine stops and when it is out of volume you could try a higher ligand concentration. If the pre-concentration rate is to high according to the algorithm you can dilute it slightly and try again until you have the optimal dilution.
I hope this will you give some clues to look at and to optimize your immobilization.