scholarly journals Electrophoretic mobility shift assay (EMSA) for detecting protein–nucleic acid interactions

2007 ◽  
Vol 2 (8) ◽  
pp. 1849-1861 ◽  
Author(s):  
Lance M Hellman ◽  
Michael G Fried
Keyword(s):  
Nucleic Acid ◽  
Electrophoretic Mobility Shift Assay ◽  
Electrophoretic Mobility ◽  
Mobility Shift ◽  
Mobility Shift Assay ◽  
Protein Nucleic Acid ◽  
Nucleic Acid Interactions

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.

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.

A fluorescence based non-radioactive electrophoretic mobility shift assay

2000 ◽  
Vol 78 (2) ◽  
pp. 163-170 ◽  
Author(s):  
K Ruscher ◽  
M Reuter ◽  
D Kupper ◽  
G Trendelenburg ◽  
U Dirnagl ◽  
...  
Keyword(s):  
Electrophoretic Mobility Shift Assay ◽  
Electrophoretic Mobility ◽  
Mobility Shift ◽  
Mobility Shift Assay

scholarly journals Human replication-dependent histone H3 genes are activated by a tandemly arranged pair of two CCAAT boxes

2004 ◽  
Vol 384 (2) ◽  
pp. 317-326 ◽  
Author(s):  
Heiner KOESSLER ◽  
Joerg KAHLE ◽  
Christa BODE ◽  
Detlef DOENECKE ◽  
Werner ALBIG
Keyword(s):  
Electrophoretic Mobility Shift Assay ◽  
Electrophoretic Mobility ◽  
Histone H3 ◽  
Maximal Activity ◽  
Significant Loss ◽  
Tata Box ◽  
Mobility Shift ◽  
Nuclear Factor Y ◽  
Binding Factor ◽  
Mobility Shift Assay

We have analysed the transcriptional regulation of the human histone H3 genes using promoter deletion series, scanning mutagenesis, specific mutagenesis and electrophoretic mobility-shift assay experiments. The promoters of five of the six examined histone H3 genes showed near-maximal activity at lengths of 133–227 bp: H3/d 198 bp, H3/h 147 bp, H3/k 133 bp, H3/m 227 bp, H3/n 140 bp (exception H3/i). To search for functional cis-elements within these regions, we performed scanning mutagenesis of the two histone H3 promoters H3/k and H3/m. Mutagenesis revealed that the functional framework of the histone H3 promoters consists of a TATA box and two tandemly arranged CCAAT boxes in relatively fixed positions. Alterations of the distance between the CCAAT boxes and of the distance between the CCAAT boxes and the TATA box resulted in significant loss of activity. In electrophoretic mobility-shift assay experiments, the factor CBF (CCAAT-binding factor)/NF-Y (nuclear factor-Y) bound to isolated CCAAT boxes of the H3/k promoter. This suggests that an initiation complex is formed on the histone H3 promoter that has a defined structure and limited flexibility, consisting of two molecules of CBF/NF-Y and further (general or specific) transcription factors.

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