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Real-Time Analysis of Specific Protein-DNA Interactions with Surface Plasmon Resonance.

Ritzefeld M, Sewald N - J Amino Acids (2012)

Bottom Line: In this article, we focus on this biosensor-based method and provide a detailed guide how SPR can be utilized to study binding of proteins to oligonucleotides.Subsequently, we will focus on the optimization of the experiment, expose pitfalls, and introduce how data should be analyzed and published.Finally, we summarize several interesting publications of the last decades dealing with protein-DNA and RNA interaction analysis by SPR.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Bielefeld University, P.O. Box 100131, 33501 Bielefeld, Germany.

ABSTRACT
Several proteins, like transcription factors, bind to certain DNA sequences, thereby regulating biochemical pathways that determine the fate of the corresponding cell. Due to these key positions, it is indispensable to analyze protein-DNA interactions and to identify their mode of action. Surface plasmon resonance is a label-free method that facilitates the elucidation of real-time kinetics of biomolecular interactions. In this article, we focus on this biosensor-based method and provide a detailed guide how SPR can be utilized to study binding of proteins to oligonucleotides. After a description of the physical phenomenon and the instrumental realization including fiber-optic-based SPR and SPR imaging, we will continue with a survey of immobilization methods. Subsequently, we will focus on the optimization of the experiment, expose pitfalls, and introduce how data should be analyzed and published. Finally, we summarize several interesting publications of the last decades dealing with protein-DNA and RNA interaction analysis by SPR.

No MeSH data available.


Related in: MedlinePlus

Typical shape of an SPR-sensogram. It can be divided into four phases: association phase, steady state or equilibrium phase, dissociation phase, and regeneration phase.
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fig2: Typical shape of an SPR-sensogram. It can be divided into four phases: association phase, steady state or equilibrium phase, dissociation phase, and regeneration phase.

Mentions: The typical shape of a sensogram that displays the change of the response units during the course of the experiment is shown in Figure 2. It can be divided into four different phases: association phase, steady-state or equilibrium phase, dissociation phase, and regeneration phase. The association phase starts with the injection of the analyte (e.g., protein). Due to the formation of a protein-DNA complex, the refractive index changes, resulting in a variation of the specific angle (Θ) where the dip in intensity of the reflected light is at its minimum. During the following equilibrium phase, association and dissociation of the complex occur at equal rates. Shortly, after the injection is terminated, dissociation of the analyte (e.g., protein) from the ligand (e.g., oligonucleotide) leads to a decrease in the response units. At any point of the dissociation phase, a regeneration buffer can be injected. It either contains a high salt concentration or detergents like SDS that release all remaining analyte molecules from the surface [12, 14]. After having finished the described cycle, another concentration of the analyte can be injected.


Real-Time Analysis of Specific Protein-DNA Interactions with Surface Plasmon Resonance.

Ritzefeld M, Sewald N - J Amino Acids (2012)

Typical shape of an SPR-sensogram. It can be divided into four phases: association phase, steady state or equilibrium phase, dissociation phase, and regeneration phase.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3303711&req=5

fig2: Typical shape of an SPR-sensogram. It can be divided into four phases: association phase, steady state or equilibrium phase, dissociation phase, and regeneration phase.
Mentions: The typical shape of a sensogram that displays the change of the response units during the course of the experiment is shown in Figure 2. It can be divided into four different phases: association phase, steady-state or equilibrium phase, dissociation phase, and regeneration phase. The association phase starts with the injection of the analyte (e.g., protein). Due to the formation of a protein-DNA complex, the refractive index changes, resulting in a variation of the specific angle (Θ) where the dip in intensity of the reflected light is at its minimum. During the following equilibrium phase, association and dissociation of the complex occur at equal rates. Shortly, after the injection is terminated, dissociation of the analyte (e.g., protein) from the ligand (e.g., oligonucleotide) leads to a decrease in the response units. At any point of the dissociation phase, a regeneration buffer can be injected. It either contains a high salt concentration or detergents like SDS that release all remaining analyte molecules from the surface [12, 14]. After having finished the described cycle, another concentration of the analyte can be injected.

Bottom Line: In this article, we focus on this biosensor-based method and provide a detailed guide how SPR can be utilized to study binding of proteins to oligonucleotides.Subsequently, we will focus on the optimization of the experiment, expose pitfalls, and introduce how data should be analyzed and published.Finally, we summarize several interesting publications of the last decades dealing with protein-DNA and RNA interaction analysis by SPR.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Bielefeld University, P.O. Box 100131, 33501 Bielefeld, Germany.

ABSTRACT
Several proteins, like transcription factors, bind to certain DNA sequences, thereby regulating biochemical pathways that determine the fate of the corresponding cell. Due to these key positions, it is indispensable to analyze protein-DNA interactions and to identify their mode of action. Surface plasmon resonance is a label-free method that facilitates the elucidation of real-time kinetics of biomolecular interactions. In this article, we focus on this biosensor-based method and provide a detailed guide how SPR can be utilized to study binding of proteins to oligonucleotides. After a description of the physical phenomenon and the instrumental realization including fiber-optic-based SPR and SPR imaging, we will continue with a survey of immobilization methods. Subsequently, we will focus on the optimization of the experiment, expose pitfalls, and introduce how data should be analyzed and published. Finally, we summarize several interesting publications of the last decades dealing with protein-DNA and RNA interaction analysis by SPR.

No MeSH data available.


Related in: MedlinePlus