This forum is intended for questions about kinetics, Surface Plasmon Resonance and the instruments related to these techniques.
New to SPR and have some questions
- gatz
- Topic Author
- Visitor
-
5 years 8 months ago #1
by gatz
New to SPR and have some questions was created by gatz
Hello,
Our lab recently purchased a P4SPR ( affiniteinstruments.com/p4spr/ ). I have taken it on myself to figure out how we can best use this machine in our lab.
We are testing peptide-based inhibitors, specifically we want to get their Kd with the protein they were designed to inhibit. We have bare-gold chips, and so far I have assembled a SAM of 16-MHDA. Our plan is to immobilize the inhibitors using amine linking (NHS/EDC) and then flow the analyte over.
My main concern is that the inhibitors are very small and quite basic. They are around 1kDa, and the pI is ~10. For pre-concentration, what buffer should I use? I see pH 6 maleic acid being used as the 'most basic' coupling buffer, but should i use a solution with a pH closer to the ligands pI, for instance a pH 9.5 buffer?
You may have noticed that the P4SPR was designed for syringe injection. We have a pump but it seems easier to just use the syringes. I know mass transport is an important factor in SPR. I'm wondering how viable it is to actually use syringes for SPR in lieu of a pump system.
I will probably have a lot more questions. Thanks in advance!
Our lab recently purchased a P4SPR ( affiniteinstruments.com/p4spr/ ). I have taken it on myself to figure out how we can best use this machine in our lab.
We are testing peptide-based inhibitors, specifically we want to get their Kd with the protein they were designed to inhibit. We have bare-gold chips, and so far I have assembled a SAM of 16-MHDA. Our plan is to immobilize the inhibitors using amine linking (NHS/EDC) and then flow the analyte over.
My main concern is that the inhibitors are very small and quite basic. They are around 1kDa, and the pI is ~10. For pre-concentration, what buffer should I use? I see pH 6 maleic acid being used as the 'most basic' coupling buffer, but should i use a solution with a pH closer to the ligands pI, for instance a pH 9.5 buffer?
You may have noticed that the P4SPR was designed for syringe injection. We have a pump but it seems easier to just use the syringes. I know mass transport is an important factor in SPR. I'm wondering how viable it is to actually use syringes for SPR in lieu of a pump system.
I will probably have a lot more questions. Thanks in advance!
Please Log in or Create an account to join the conversation.
- Arnoud
- Visitor
-
5 years 8 months ago #2
by Arnoud
Replied by Arnoud on topic New to SPR and have some questions
Hi,
Amine coupling generally uses the amine on the Lysine residue and not the terminal NH2 of the peptide. Thus is your peptide suitable for the coupling?
You could try a pre-concentration experiment ( www.sprpages.nl/immobilization/ligand-pre-concentration ) at different pH-values but I am not sure this will be efficient on a planar surface. The pH recommendations are normally based on the pre-concentration effect on carboxy-dextran based sensor surfaces. I your case I don't think it matters that much.
Mass-transport is only a concern when you have much ligand/many binding places on the sensor chip. You can calculate the amount of ligand to be immobilised to get a proper signal ( www.sprpages.nl/immobilization/immobilization-procedures ). In Biacore terms this would be around 100-200 RU and at these values mass-transport is generally very low.
I have no experience with syringe operated instruments but a constant flow would be essential to get nice association and dissociation curves.
Kind regards
Arnoud
Amine coupling generally uses the amine on the Lysine residue and not the terminal NH2 of the peptide. Thus is your peptide suitable for the coupling?
You could try a pre-concentration experiment ( www.sprpages.nl/immobilization/ligand-pre-concentration ) at different pH-values but I am not sure this will be efficient on a planar surface. The pH recommendations are normally based on the pre-concentration effect on carboxy-dextran based sensor surfaces. I your case I don't think it matters that much.
Mass-transport is only a concern when you have much ligand/many binding places on the sensor chip. You can calculate the amount of ligand to be immobilised to get a proper signal ( www.sprpages.nl/immobilization/immobilization-procedures ). In Biacore terms this would be around 100-200 RU and at these values mass-transport is generally very low.
I have no experience with syringe operated instruments but a constant flow would be essential to get nice association and dissociation curves.
Kind regards
Arnoud
Please Log in or Create an account to join the conversation.
Moderators: Arnoud, Arnoud