scholarly journals Practical strategies for the evaluation of high-affinity protein/nucleic acid interactions

2013 ◽  
Vol 4 (1) ◽  
pp. 3 ◽  
Author(s):  
Sarah E. Altschuler ◽  
Karen A. Lewis ◽  
Deborah S. Wuttke
Keyword(s):  
Nucleic Acid ◽  
Binding Affinities ◽  
Mobility Shift ◽  
Experimental Conditions ◽  
High Affinity ◽  
Apparent Binding Constant ◽  
Highly Sensitive ◽  
Mobility Shift Assay ◽  
Protein Nucleic Acid ◽  
Nucleic Acid Interactions

The quantitative evaluation of binding interactions between proteins and nucleic acids is highly sensitive to a variety of experimental conditions. Optimization of these conditions is critical for obtaining high quality, reproducible data, particularly in the context of very high affinity interactions. Here, we discuss the practical considerations involved in optimizing the apparent binding constant of an interaction as measured by two common quantitative assays, electrophoretic mobility shift assay and double-filter binding when measuring extremely tight protein/nucleic acid interactions with sub-nanomolar binding affinities. We include specific examples from two telomere end-binding protein systems, <em>Schizosaccharomyces pombe</em> Pot1 and <em>Saccharomyces cerevisiae </em>Cdc13, to demonstrate potential experimental pitfalls and some useful strategies for optimization.

Snapshot blotting: The transfer of nucleic acids and nucleoprotein complexes from electrophoresis gels to EM grids

1992 ◽  
Vol 50 (1) ◽  
pp. 762-763
Author(s):  
Stephen D. Jett
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Nucleic Acid Binding ◽  
Mobility Shift ◽  
Carbon Coated ◽  
Nucleoprotein Complexes ◽  
Mobility Shift Assay ◽  
Protein Nucleic Acid ◽  
Gel Mobility Shift ◽  
Nucleic Acid Interactions

The electrophoresis gel mobility shift assay is a popular method for the study of protein-nucleic acid interactions. The binding of proteins to DNA is characterized by a reduction in the electrophoretic mobility of the nucleic acid. Binding affinity, stoichiometry, and kinetics can be obtained from such assays; however, it is often desirable to image the various species in the gel bands using TEM. Present methods for isolation of nucleoproteins from gel bands are inefficient and often destroy the native structure of the complexes. We have developed a technique, called “snapshot blotting,” by which nucleic acids and nucleoprotein complexes in electrophoresis gels can be electrophoretically transferred directly onto carbon-coated grids for TEM imaging.

Use of difference boundary sedimentation velocity to investigate nonspecific protein-nucleic acid interactions

Biochemistry ◽  
1980 ◽  
Vol 19 (15) ◽  
pp. 3516-3522 ◽  
Author(s):  
Timothy M. Lohman ◽  
C. Glen Wensley ◽  
Jeffrey Cina ◽  
Richard R. Burgess ◽  
M. Thomas Record
Keyword(s):  
Nucleic Acid ◽  
Sedimentation Velocity ◽  
Protein Nucleic Acid ◽  
Nucleic Acid Interactions

Protein—nucleic acid interactions Folding and binding Web alert

1998 ◽  
Vol 8 (1) ◽  
pp. 9-10 ◽  
Author(s):  
PhilipE Bourne ◽  
Judith Murray-Rust ◽  
JeremyH Lakey
Keyword(s):  
Nucleic Acid ◽  
Protein Nucleic Acid ◽  
Nucleic Acid Interactions

Nonspecific Prion Protein–Nucleic Acid Interactions Lead to Different Aggregates and Cytotoxic Species

Biochemistry ◽  
2012 ◽  
Vol 51 (27) ◽  
pp. 5402-5413 ◽  
Author(s):  
Bruno Macedo ◽  
Thiago A. Millen ◽  
Carolina A. C. A. Braga ◽  
Mariana P. B. Gomes ◽  
Priscila S. Ferreira ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Prion Protein ◽  
Protein Nucleic Acid ◽  
Nucleic Acid Interactions
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