12AA - amino acids? It is just too small (1.2-1.5 kDa) for many SPR devices. Take into account that even in the ideal case (the dense monolayer of much larger receptor proteins) the surface coverage by bound peptide will be much lower than monolayer (just because receptor protein takes more place on the surface).

So depending on what instrument you are actually using, whether the receptor is immobilized as monolayer or in the matrix, you might be just beyond limits of detection.  

I am not sure whether it is possible to immobilize peptide and let protein bind to it, but that would result in much stronger  signal.

There may be also concerns regarding that the immobilization of protein on the surface of the SPR sensor may influence its ability to bind antibody. It is a known disadvantage of SPR biosensing, but most probably you are not dealing with such case.

Hi Shavkat

Thanks for the answer

The 38kDa protein was immobilized and not the peptide.

 Hi Pavel

The buffers that were used for the protein-peptide interaction were PBS/BSA;

This was the protocol used for the SPR

Binding experiments were performed on Biacore T-200 at 25°C.  The ligands 38kDa proteins were directly immobilized by amine coupling (EDC/NHS) at an RU (Response units) of 2000, 3000. The un-occupied sites were blocked with 1M ethanol amine.   The analyte as indicated was flowed over the chip at variable concentrations.  

Hi Avner,

In our experience that should not be too small to measure on most SPR instruments. The more important parameter is the ratio of the molecular weights of the analyte and ligand. For your case, 1200Da/38,000DA = 0.032. So the amount of response you can expect for perfect 1:1 binding is 0.032 * amount of ligand immobilized. So for example if you immobilized 1000 RU of ligand you should expect 32 RU max response from your analyte. You can use this to estimate if you are below the detection limit of the instrument based on how much ligand you have immobilized.  

If you are in the detectable range and still not seeing any binding, you could try changing the density of ligand (higher and lower), to see if this has any impact. Definitely trying other types of immobilization is another thing we would suggest. You could also try changing buffer conditions. Maybe there is some kind of charge repulsion between your analyte and the chip surface, so adding more salt might help, just as an example.  

Hi, peptides binding to a protein is pretty much possible using SPR an there are lots of literature available on this. All you need to to do is design the assay properly. Select the correct sensorchip, you can use a sensorchip CM3 (if biacore-SPR system) for attacing the portein (as ligand), immobilize close to 3000 to 5000RU and then use peptides as analyte. in this approach, you should be aware of the regeneration conditions, running and immobilization buffer conditions suitable for the assay and retaining the ligand activity as well. another approach, is you can biotinylate your peptide and use neutravidin chip (sensorchip NA) or streptavidin chip (Sensorchip SA) to attach peptide to the sensor surface and pass protein as analyte. This approach will be much more simpler and easier. you need any help, you can write to me directly.