I want to investigate FcRn-Albumin interaction by SPR. my first try was done by immobilization FcRn on a CM5 sensor chip in pH 1 unit lower than pI of FcRn in sodium acetate and I get a strange sensogram and I couldn't measure my kinetic also. I think I had a mistake in the immobilization procedure. Based on chemistry, activation of carboxymethyl surface by NHS/EDC should be done at pH 4.5-7.2, and the reaction of NHS-activated molecules with primary amines of the ligand is most efficient in pH7-8 (Ref: Thermo scientific, attached file), but in the SPR instrument book (Biocare) mention ligand immobilization by amine coupling should be done at pH 1 unit lower than the isoelectric point of ligand due to electrostatic interaction toward the carboxymethylated surface. I get confused! which pH should be set for ligand immobilization?
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You can test the best immobilization pH with an pre-concentration experiment.
Activate the surface with a mix of NHS/EDC and directly inject the ligand to be immobilized. Don't forget to deactivate with ehanolamine to quech not used activated sites.
thanks for your reply dear Around.
yeah, I did preconcentration and pH 4.5 was the most suitable one.
I have some question;
1) before immobilization, equilibration of the system by which buffer should be done?! I applied HEPES which my analyte dissolve in it.it shouldn't be done by a buffer which EDC and NHS dissolved in?!
2)in which buffer EDC/NHS should be dissolved?
3) I attach my immobilization sensorgram, why the peak after ligand injection goes down?! a window appears on the web showed me near 700 RU immobilized FcRn, but the graph doesn't show this amount?!
my advisor suggests me to follow Thermo science protocol, they applied MES buffer before ligand injection and ligand dissolve in phosphate buffer by pH 7-8! I have doubt it works!? why Thermo doesn't mention about any electrostatic interaction?! I will be happy if you can give me any suggestion
Thank you very much.
1) equilibrate the system with HBS pH 7.4-7.5 or another standard buffer without primary amines.
2) NHS and EDC should be dissolved in pure water. Excess solution can be aliquoted and stored frozen at -20°C. Once thawed use directly and don't refreeze. (
3) not sure what you mean. In the sensorgram I see the pre-concentration step, activation step, four ligand injections and deactivation step.
The ligand injection steps go down because of the low salt content of the sodium acetate compared to the flow buffer. In addition you see the curves go upwards during the injection which means that the pre-concentration works. The first ligand injection (pre-concentration test) you see that as soon as the injection stops the curve returns almost to base line. That is good. Normally you proceed with an injection of 50 mM NaOH to remove all electrostatic bound material before the activation of the surface.
Activation of the surface is done with a 1:1 mix of 0.4 M EDS: 0.1 M NHS for seven minutes and should give a baseline rise of 100 – 200 RU.
In the sensorgram I see that the ligand is binding to the surface since the baseline after the injections is higher (700 RU?) than before the injections. In addition after deactivation the 'new ligand' baseline is the same. My conclusion is that something is immobilized on the surface.
>why Thermo doesn't mention about any electrostatic interaction?!
The electrostatic interaction is something specific for carboxylated (dextran) surfaces. The activation step only activates a part of the carboxyl groups to N-Hydroxysuccimide ester and therefore during the ligand injection at low pH the remaining carboxyl groups have a negative charge. The ligand (protein) should have a positive charge (pH solution < pI ligand) and therefore the ligand is drawn to the sensor surface which provides a high local ligand concentration and efficient immobilization chemistry. The chemical reaction itself is not dependent on the pre-concentration and will occur between pH 4 – 6 (7.2).