SPR Non-Specific Binding

Hello,
I would to get your advice regarding the possible reasons for non-specific binding with my SPR experiment.
The Experimental condition is as the following;
1- Legend ssDNA Aptamer (500RU).
2- Analyte Protein (500nM), PI 5.4.
3- Buffer; HBS-EP
4- FC1 &FC2 immobilized with PolyA 20N.
5- FC2 immobilized with Aptamer containing PolyT 20N.
6- Machine SPR X100

Other points, I noticed 2 non-specific peaks at 70sec and 250sec, would you let me what is the reason behind these peaks?

Thank you in Advance.
Waleed

Hi Waleed,
Assuming that the red line is the reference and the green the active surface you can see that in both the buk shift upon injection is quite high. Subtracting the reference wil not compensate fully and because both channels are in series will give the spike at the begin and end of the sensorgram. The only remedy is to match sample and running buffer better ( www.sprpages.nl/best-results/artifacts/spikes ).

Non-specific binding can have many origins. You do not mention if the non-specific binding is to the reference alone. If so, my suggestion is to block the reference with a rondom DNA-polyT sample or block the polyA with poly-T. ( www.sprpages.nl/best-results/artifacts/non-specific ).

kind regards
Arnoud

Dear Arnoud
Thank you for your comment.
The non-specific spike appears in both channels. In addition, I diluted the analyte protein in the running buffer (1:30).
In this experiment I have used the hybridization approach as the following;
1- Legend ssDNA Aptamer (500RU Immobilization).
2- Analyte Protein (500nM), diluted in the running buffer, PI 5.4.
3- Buffer; HBS-EP
4- Both channels (FC1 &FC2) are immobilized with PolyA 20N.
5- FC2 immobilized with Aptamer containing PolyT 20N. It is also noted the the the buffer control sample doesn't show non-specific binding in both channels.
DO you think that there is a possibility of non-specific interaction between protein and immobilized PolyA?

Thank you in advance.
Waleed

Hi Waleed,

Still for my understanding:
What kind of chip do you use to immobilise the polyA to (and how) in FC1&2?
Then you flow the polyT-aptamer over the FC2.
Then flow the analyte over FC1&2.

Injecting buffer alone gives no spikes at the begin and end of injection?
Injecting buffer alone gives no bulk jump and a flat line?

Non-specific binding can have many origins even the bare sensor surface can bind something. Only careful experimentation can identify the type of non-specific binding and give clues to avoid or minimize.

Kind regards
Arnoud

Thank You, Arnoud for continuous help.
I have used SA chip, immobilize the polyA to FC1&2 and polyT-aptamer over the FC2 only. then, run the analyte over FC1&2.
Injection buffer alone gives no spikes and I think good curve as a control.
I have more diluted the analyte in the running buffer (1:250) to avoid Bulk Response
. However, I still have the same problem.
I plan to make a buffer exchange for the analyte (with Running buffer).
Please let me know if you have other advice.

Best Wishes,
Waleed