FAQ-List


How much ligand do I need ?
For an average immobilization you need 25 µg of ligand.


How much analyte do I need ?
This depends on the kinetics between ligand and analyte. For kinetic studies a range of concentrations between 0.01 - 100 x KD is injected. If possible the injection should be long enough to reach steady state. For a two minute injection with an antibody at 50 nM at 25 µl/min 0.75 µg of antibody in needed. 100 µl of a stock solution of 5 µM (0.75 µg/ml) antibody is sufficient to do the first kinetic experiments.


How long does it take to immobilize a ligand on the chip?
Starting from scratch it can take between 45 – 90 minutes per channel. When the immobilization conditions have to be established by a pre-concentration experiment it can take some extra time. When immobilization conditions are known a simple amino coupling can be done within 30 minutes.


How much ligand must I immobilize ?
The amount of ligand to be immobilized depends on the application.

  • For specificity measurements, almost any ligand density will do as long as it gives a good signal.
  • Concentration measurements need the highest ligand density to facilitate mass transfer limitation.
  • Affinity ranking can be done with low to moderate density surfaces as long as the analyte can saturate the ligand.
  • Kinetics are done with the lowest ligand density possible, giving a proper signal.

For more information goto immobilization page.


Which coupling buffer should I use ?
For the standard amine-coupling a low salt buffer (e.g. 10 mM Na-acetate, pH 3.0 - 5.0) without reactive components (e.g. TRIS, azide) is recommended.


Is it possible to measure conformational change with SPR ?
It is not likely you can. Since conformation change does not change the signal but the kinetics, it can be difficult to confirm real conformational change. In addition look at the Conformational change page.


What chemistry should I use to modify my ligand ?
Depending on your ligand and sensor chip several possibilities are available. Look at the immobilization page for an overview of the common used chemistries. When there is a difficult ligand then turn to Pierce for more possibilities.


How to set up an efficient regeneration ?
The regeneration procedure has to be evaluated emperically because the physical forces responsible for the binding are often unknown. Therefore, no single regeneration solution can be recommended. For more information goto the regeneration page.


Is it difficult to establish an assay for DNA-protein and protein-protein interactions?

The more information you begin with the better. As with any technique, you must understand what you doing before you get meaningfull results. Start with reading.

(1) Burgener, M. et al Synthesis of a stable and specific surface plasmon resonance biosensor surface employing covalently immobilized peptide nucleic acids. Bioconjugate Chemistry 11: 749-754; (2000).
(2) Ciolkowski, M. L. et al A surface plasmon resonance method for detecting multiple modes of DNA-ligand interactions. J.Pharm.Biomed.Anal. 22: 1037-1045; (2000).
(3) Hao, D. et al A modified sensor chip for surface plasmon resonance enables a rapid determination of sequence specificity of DNA-binding proteins. FEBS Letters 536: 151-156; (2003).
(4) Jensen, K. K. et al Kinetics for hybridization of peptide nucleic acids (PNA) with DNA and RNA studied with the BIAcore technique. Biochemistry 36: 5072-5077; (1997).
(5) Nilsson, P. et al Real-time monitoring of DNA manipulations using biosensor technology. Analytical Biochemistry 224: 400-408; (1995).
(6) Oda, M. and Nakamura, H. Thermodynamic and kinetic analyses for understanding sequence-specific DNA recognition. Genes Cells 5: 319-326; (2000).


Is it very difficult to study ligand-analyte interaction within a lipid bilayer?
No. Biacore has some specialized sensor chips like HPA, which mimics a lipid surface, or L1 on which a lipid bilayer can deposited.

Some HPA sensor chip references are:

(1) Bader, B. et al Bioorganic synthesis of lipid-modified proteins for the study of signal transduction. Nature 403: 223-226; (2000).
(2) Cooper, M. A. et al Binding of vancomycin group antibiotics to D-alanine and D-lactate presenting self-assembled monolayers. Bioorg.Med.Chem. 8: 2609-2616; (2000).
(3) Hodgkin, M. N. et al Phospholipase D regulation and localisation is dependent upon a phosphatidylinositol 4,5-biphosphate-specific PH domain. Curr.Biol. 10: 43-46; (2000).
(4) Isawa, H. et al The insect salivary protein, prolixin-S, inhibits factor IXa generation and Xase complex formation in the blood coagulation pathway. J.Biol.Chem. 275: 6636-6641; (2000).
(5) Satoh, A. et al Ligand-Binding Properties of Annexin from Caenorhabditis elegans (Annexin XVI, Nex-1). Journal of Biochemistry 128: 377-381; (2000).
(6) Pommier, J. et al TorD, a cytoplasmic chaperone that interacts with the unfolded trimethylamine N-oxide reductase enzyme (TorA) in Escherichia coli. J.Biol.Chem. 273: 16615-16620; (1998).

Some L1 sensor chip references are:

(1) Bitto, E. et al Mechanism of annexin I-mediated membrane aggregation. Biochemistry 39: 13469-13477; (2000).
(2) Cooper, M. A. et al A vesicle capture sensor chip for kemitic analysis of interactions with membrane bound recptors. Anal.Biochem 277: 196-205; (2000).
(3) Danelian, E. et al SPR biosensor studies of the direct interaction between 27 drugs and a liposome surface: correlation with fraction absorbed in humans. J.Med.Chem. 43: 2083-2086; (2000).
(4) Erb, E. M. et al Characterization of the surfaces generated by liposome binding to the modified dextran matrix of a surface plasmon resonance sensor chip. Analytical Biochemistry 280: 29-35; (2000).
(5) Cooper, M. A. et al Binding of vancomycin group antibiotics to D-alanine and D-lactate presenting self-assembled monolayers. Bioorg.Med.Chem. 8: 2609-2616; (2000).
(6) Hodgkin, M. N. et al Phospholipase D regulation and localisation is dependent upon a phosphatidylinositol 4,5-biphosphate-specific PH domain. Curr.Biol. 10: 43-46; (2000).
(7) Isawa, H. et al The insect salivary protein, prolixin-S, inhibits factor IXa generation and Xase complex formation in the blood coagulation pathway. J.Biol.Chem. 275: 6636-6641; (2000).
(8) Satoh, A. et al Ligand-Binding Properties of Annexin from Caenorhabditis elegans (Annexin XVI, Nex-1). Journal of Biochemistry 128: 377-381; (2000).
(9) Pommier, J. et al TorD, a cytoplasmic chaperone that interacts with the unfolded trimethylamine N-oxide reductase enzyme (TorA) in Escherichia coli. J.Biol.Chem. 273: 16615-16620; (1998).


Is it possible to remove the bound ligand ?
Is it possible to remove the bound ligand and to immobilize a different ligand on the same senor-chip without any lost of performance? There is one publication about this.

(1) Chatelier, R. C. et al A general method to recondition and reuse BIAcore sensor chips fouled with covalently immobilized protein/peptide. Analytical Biochemistry 229: 112-118; (1995).