|Capture Kinetic||Direct Kinetic|
In order to prevent mass transport limitation as influencing parameter on the kinetics, a low-medium response level for the solution sample should be aimed for. As a good starting point, try to achieve max. 20-60 RU for your solution sample or antigen. Calculate your maximum solution sample response using equation (6-1) [https://www.sprpages.nl/sensorgram-tutorial/a-curve].
Especially doing capture kinetics the surface sample density can be controlled efficiently, by performing test capture/binding and find an optimum. Start with initial capture injection times of 15-45 s and start with 0.25-1 ug/mL of your molecule to be captured.
In general start with gentle conditions and short contact times, if a new regeneration condition has to be evaluated. After a successful regeneration retest the binding and proof the activity of your capture molecule.
Proper regeneration conditions:
|Strength||Type of bond|
|Weak||ph > 2.5||ph < 9||ph < 9|
|10mM glycine/HCl||10mM HEPES/NaOH||25–50% ethylene glycol||0.5–1 M NaCl|
|1–10 mM HCl||0.02% SDS|
|0.5M formic acid|
|Intermediate||ph 2 – 2.5||ph 9 – 10||ph 9 – 10|
|0.5M formic acid||10–100mM NaOH||50% ethylene glycol||1–2M MgCl2|
|10mM Glycine/HCl||10mM Glycine/NaOH||0.5–0.5% SDS||1–2M NaCl|
|Strong||ph < 2||ph > 10||ph > 10|
|1M formic acid||50–100mM NaOH||25–50% ethylene glycol||2–4M MgCl2|
|10–100mM HCl||6M guanidinechloride||0.5% SDS|
|10–50mM Glycine/HCl||1M ethanolamine|
|0.1% trifluoracetic acid|
Raising temperature, may speed up the off-rate
Increasing the dissociation time
In order to get qualified information from an off-rate, a certain decay needs to be detected. At a dissociation time of 180-600s it is difficult to see differences for off-rates at 10-4 and 10-5 s-1 . Thus a longer dissociation time should be chosen.