Streptavidin capturing and small molecules

Hi all,

I am trying to use streptavidin capturing method for testing binding of small molecules to Ligand target.

I used CM5 to immobilize streptavidin, with a level of immobilization of about 10,000 then when I tried to capture the biotinylated protein, the maximum level I got was 2700 (the protein was of about 28,000-30,000 D). However when I am trying to test the binding of a small molecule to that captured protein I am getting non concentration-dependent response from different concentrations. Any idea what is causing that?

I forgot to mention that the same Ligand protein I have used with direct immobilization and got good results with the same small molecule.

Quick question: did you purify the biotinylated protein to get rid of the unbound biotin?

I have tried two methods for purification, first I tried Zeba desalting column from ThermoFisher then I have used dialysis which gave me better purification from free biotin

Hi Ahmed,

I ahve no idea what could cause this. Perhaps these two publication can help you.

1. Papalia, G. and Myszka, D.; Exploring minimal biotinylation conditions for biosensor analysis using capture chips. Analytical Biochemistry (403) 1-2: 30-35; 2010.
2. Peter, J. C., Briand, J. P. and Hoebeke, J.; How biotinylation can interfere with recognition: a surface plasmon resonance study of peptide-antibody interactions. Journal of Immunological Methods (274) 1-2: 149-158; 2003.



Arnoud

Hi Arnoud,

Thanks a lot for your reply.

I was wondering if you have used this method of immobilization of streptavidin on CM5 and capturing biotinylated protein to test the binding of small molecules; as I found some publication using this method to capture DNA and testing the binding with small molecules but not protein.

The first publication you sent to me is using the reusable capture chip with CA II, although the guide of capture chip saying it should not be used with protein less than 150KD and CA II is about 30KD, which I think that is why they have not tried to use this method to test the binding of the captured CA II with small molecules inhibitors.

So do you think this method is suitable at all for small molecules specially using BIAcore 3000.

Thanks again

Hi Ahmed,

I tried to find a protocol to immobilize the streptavidin to the sensor chip but could not find one yet. I did use the CAP-system of Biacore and it performed very well. The added advantage is, that the chip is reusable since you can strip the streptavidin-biotin-protein complex.
You can also buy the SA-chip which is probably a little more expensive but of good quality.

You can try to calculate if the compound is not too small for the system ( www.sprpages.nl/immobilization/immobilization-strategy ).

I can measure the acetazolamide (222 Da)over a CAII surface (29 kDa; 4000 RU) which will give something between 12 and 20 RU on a Biacore 3000.

I hope you will get your system working.

Kind regards

Arnoud