No binding seen - Biotin. oligo and Ab

Hello. I was trying this experiment. Details as follows. Two reagent. A) Biotinylated Oligo (6500 Da), Ab (150kDa), I immobilized the Biotin-Oligo unto a streptavidin chip (GE) at 3 different levels. FC 1 used as blank, no injections. Performed manual injections for this step. I then passed on the Ab on all FC's 1 conc. only (50nM). I am not seeing any binding. I used HBS-EP buffer and used the same diluent for all dilutions. Other info are: Before immobilization, SA surface was pre-conditioned with 0.5M NaCL / 50mM NaOH at 50uL/min for 5 mins. Performed 2x. Then after the Biotin-Oligo was immobilized, performed a blocking step to block other unoccupied SA sites with 50uM amino-PEG using a flow rate of 30uL/min for 2 min. Any suggestions on how I can obtain binding between the 2. 6 sensorgrams are attached. 3 for the immobilization and 3 for the Ab. Does Any one have any experience in doing the experiment above? Thank you.

Hi,
From the sensorgrams I conclude that the oligo is bound to the sensor chip. At what level (RU)?
I assume that the oligi-sequence is the recognition site for the antibody. Did you validate the binding between oligo and antibody in an other set-up? ELISA?

Kind regards
Arnoud

Hi Arnoud, Immobil levels are: FC2: 538, FC3: 11, FC: 102. I tried at 3 different levels hoping that I would see one that will show binding. Submitter informed me that they did do an orthogonal test (need to determine what technique), and they did say that binding was observed, although at much higher concentration that what I used for the experiment. Thanks.