Surface Plasmon Resonance - Molecule immobilization (Jan/03/2008 )

Hi all,

what is the real immobilization level if you use amine coupling?

1: level after ethanolamine injection - level before surface activation?

or

2: level after protein injection - level before protein injection?

Thanks

hi celvas,

it depends on which protein you inject and at which concentration
in general, amine coupling permits to immobilize ca 15,000 RU of a first layer of antibody at 30 µg/mL
EDC/NHS activation do not increase the signal significantly
I have often observed a 20,000 RU increase after the protein injection and before CM5 inactivation
you may lose 5,000 RU after ethanolamine injection
and even a little beat more after some washes (HCl 50mM 3-5 times for example), which eliminate non-covalently but tightly bound protein molecules

Sebastien

Thanks for your reply Tryptofan,

but, as you know, depending on the goal of the experiment you may need
to immobilize a low amount of protein, below 100 RU.

In these cases, how do you calculate the immobilization level?

It is not easy to control the level of protein to immolize at this stage...
And I am not an expert for immobilization of small amount of ligand!

In your case, I would use a highly diluted solution of the protein
Inject the different compounds manually and stop when obtaining 5,200 RU after injection of the protein (5,000 RU for the loss after inactivation and ca 100 RU for the loss after washes)

Sebastien