Forum :: Streptavidin immobilisation

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TOPIC: Streptavidin immobilisation

Streptavidin immobilisation 24 Feb 2012 01:00 #1

I'm an Italian student and I started working with Biacore few months ago, so I'm a beginner.
I immobilized streptavidin on CM5 chip with this immobilization procedure :

Flow: 5µl/min
Running buffer: PBS 100mM + NaCl 200mM + TWEEN 0,01% + BSA 0,2mg/ml

1) Activation: NHS+EDAC 7min, 5µl/min
2) Coupling: Streptavidin 200ppm, acetate buffer 10mM pH5, 15min, 5µl/min
3) Deactivation: ethanolamine pH8, 7min, 5µl/min

My problem is this one: I don’t get always the same result. Usually the curve is characterized by a drop during the streptavidin injection phase (point 2). Only sometimes I get a good immobilization curve.
Below two different sensorgrams: I used the SAME buffers and the same immobilization procedure in both cases.
I’ve this trouble only with streptavidin. No problems with Ab or other proteins immobilization. I tried to change streptavidin stock and to prepare new buffers too, but this has not solved the problem.

I’m sorry for my English, I hope it was clear.
Thanks in advance for your help!


Last Edit: 22 Dec 2013 10:10 by OldForum.

Streptavidin immobilisation 24 Feb 2012 01:00 #2

It looks like that something is mixing with the streptavidin.
Use a inject to minimize mixing and check visually that the air plugs are in place.
Wash after activation may help.
Otherwise, run systemcheck to verify all the valls are properly working.

Streptavidin immobilisation 24 Feb 2012 01:00 #3

Why do you use BSA in the buffer during immobilisation? I would try without it and put it later in the buffer if you are trying to lower the non-specific binfing.
Have you tried shorter pulses?
It could be that microfluidic system is clogged partialy and that injections are not reliable?
And ofcourse as a Biacore user you can use SA chips which are coated with streptavidin.