Determination of the best concentration range for SPR experiments

Hello!

I am currently working with SPR in order to determine peptides constant of dissociation (KD) and Rmax. My problem is how to find a good concentration range without working a lot of experiments? I found a document in SPR Software part on this page (nammed KD assistant introduction), but I don't understand very well how it works. Can I have other documents to help me? Could you please explain to me how I can choose the concentration of the analyte to be tested manually in order to obtain software input parameters? Where can I find the software for Mac IOS ?

The other question which is not related to the above question is how I can find Rmax, knowing only the KD (from the literature for example if they only give KD and you want also Rmax to do some studies).

Best Regards!

Hi Rachel,

KD Assistant: unfortunately there is no MAC IOS version (and not planned).
First you choose of dissociation curve observed from you test injection.
As from figure 7 in the KD Assistant introduction you determine the initial slope (RU/s) from a test injection, plus the response (RU) after three minutes (baseline = zero) and the concentration of the used analyte (nM). Hit calculate and the program gives the highest analyte concentration. The make a step three dilution with six samples and inject.

If you know only the K_D there is a problem that you don't know how it is composed from k_a and k_d. In my opinion reporting only the K_D is a very bad practice. The Rmax is not related to the K_D or kinetics. The Rmax is used to scale the fitting and can be predicted when you know how much ligand you immobilized and know the size ratio between ligand and analyte. See the 'Curve dependencies' figure at www.sprpages.nl/sensorgram-tutorial/a-curve



To measure the Rmax of an interaction pair you have to saturate the ligand with analyte. This means that you need at least 10 times the K_D and a long enough association period. In general, the fitting procedure will calculate the theoretical Rmax for you. For the calculation to be accurate you need at least one analyte concentration above the K_D and enough curvature in the lines.
I can also suggest the SPRsimulation to play around with kinetic parameters and you also support the SPRpages with it.

Kind regards
Arnoud

( www.sprpages.nl/sensorgram-tutorial )

can you please elaborate the annotations as this are confusing to me
Mr(analyte) : molecular weight of analyte
Mr (Ligand) : molecular weight of protein
Valency (Ligand): stoichiometry value
R(ligand): ???
Please correct me if i am wrong

Mr(analyte) : molecular weight of analyte
Mr (Ligand) : molecular weight of protein = bound to sensor surface
Valency (Ligand): stoichiometry value
R(ligand): ??? = amount of ligand bound to sensor surface

Basically the formula calculates the theoretical response when all the immobilized ligand is saturated with the analyte.

Arnoud

thank you,
the theoretical Rmax of analyte at particular concentration???
so thats what i want to know or this question asks. where all the annotations in formula are correct as i have mention in previous comment. and also please let us know the what is R(ligand)???

R(ligand): is the amount of ligand bound to sensor surface during immobilization
Rmax is the maximal response that can be measured. it is independent of analyte concentration.
Please read the website (kinetics and Sensorgrams) for more information,

Arnoud