Limits...
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.


Illustration of the ribonuclease H reaction, involving (a) mass transport, (b) enzymatic adsorption, and (c) hydrolysis [89].
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3303711&req=5

fig8: Illustration of the ribonuclease H reaction, involving (a) mass transport, (b) enzymatic adsorption, and (c) hydrolysis [89].

Mentions: (17)E(x=∞)→km  E(x=0)+S⇌kdkaES→kcat  S∗+E(x=0),where E(x=∞) and E(x=0) are the bulk and surface enzyme species, respectively, km is the corresponding mass transport coefficient, S the RNA-DNA heteroduplex, ES the enzyme-substrate complex, and S* the reaction product (single stranded DNA). A different illustration of the reaction scheme is presented in Figure 8.


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

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

Illustration of the ribonuclease H reaction, involving (a) mass transport, (b) enzymatic adsorption, and (c) hydrolysis [89].
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig8: Illustration of the ribonuclease H reaction, involving (a) mass transport, (b) enzymatic adsorption, and (c) hydrolysis [89].
Mentions: (17)E(x=∞)→km  E(x=0)+S⇌kdkaES→kcat  S∗+E(x=0),where E(x=∞) and E(x=0) are the bulk and surface enzyme species, respectively, km is the corresponding mass transport coefficient, S the RNA-DNA heteroduplex, ES the enzyme-substrate complex, and S* the reaction product (single stranded DNA). A different illustration of the reaction scheme is presented in Figure 8.

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.