SPR Rookie. Curious spikes and line crossing

Thanks in advance for considering my question. I am very new to SPR and am trying to digest all the information for setting up and achieving high-quality data. Some quick information: I'm using a T100, CM5 chip with protein covalently attached (FC2_2,500RUs, FC4_10,000 RUs FC1 and 2 are blocked with ethanolamine). My analytes are carbohydrates with low protein interactions. I'm using PBS-N as my running buffer and using Fc4 (10,000 RUs). The machine has been cleaned several times using the desorb and sanitize protocols in addition to the flow cell cleaning using the 2-propanol / NaOH cocktail. Running solvents are prepared fresh and degassed (in addition a new degasser unit was installed) I've noticed that some sensogram data are quite nice (See attached) with smooth line even at high concentrations 3mM. However, this other analyte produces annoying peaks at injection and after contact time expires. Note, I run 10 blank runs prior to starting my analyte experiments and I use the Scrubber 2.0 software to subtract blank run. (see attached) I have attached three files that illustrate the good data, my blank runs having fairly consistent outcomes, and my trace of interest that is a bit perplexing. Any suggestions are appreciated and thank you in advance.

Hi and welcome to the SPR community.

I think you are well underway. I think that the 10x blanks runs are a bit overdone. We normally have three and do some blank runs (zero analyte concentration) in between the analyte injections (once every 4-5 injections).
The blanks look ok and what you see is the noise of the instrument and is in the normal range. The curves of the first sensor gram look better because of the scale. When you zoom in you will see the same noise level as with the blanks, which is ok.
About the first sensorgram: this looks like a fast-on, fast on profile. You can make an equilibrium plot to find out if your analyte concentration is already high enough to do some fittings. You have to make sure that all bulk jumps are properly removed by referencing (see below).
What you see at the third sensorgram is probably coming from non-optimal references curves. The jumps at the injection start and –end are introduced when you subtract the reference from the active channel. This is the case when the bulk effect on the reference is bigger or smaller than the effect on the active channel. You can try the 'Jump-option' in scrubber (Reference tab) to minimize this.

I suggest you read www.sprpages.nl/sensorgram-tutorial and maybe buy the SPR-pages book.

Kind regards
Arnoud

Arnoud, Much appreciation for the thorough response.

1. The first sensogram is definitely a fast-off system with a miserable KD. The affinity plot (RUs vs. conc) has not hit the inflection point, even at 3mM. To be honest, I was using that compound to test the system.
2. In the future I will reduce the number of start-up runs and incorporate several blanks (zero concentration of the analyte) during the run.
3. I will look into the 'Jump' function on the Scrubber software to see if this provides any help. I'm surprised that there is such a large difference bulk effect between two different analytes, especially when the analyte in the third sensogram is at a lower concentration.

Finally, I am interested in buying the SPR book. Is it available to those of us in the United States? If not, I'll have a collaborator in the Netherlands buy it and send it to me.

Thank you again!

Hi Anthony,
You can buy the book via the website at www.sprpages.nl/contact-spr-pages/sprpages-book
I will be send to the US if you wish.

Arnoud