Improving Sensitivity of the Electrophoretic Mobility Shift Assay by Restricting Tissue Phosphatase Activities

Laniel, Marc-André; Guérin, Sylvain L.

doi:10.2144/98246st02

Cited by 4 publications

References 10 publications

(16 reference statements)

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Protein–Oligonucleotide Interactions

Gabrielsen

Matre

Bergholtz

2000

Encyclopedia of Analytical Chemistry

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The specific binding of a protein to a nucleic acid is a first step in several central processes in a living cell. Sequence‐specific protein–DNA interactions are crucial for the functional read‐out of genetic information. Sequence recognition is the result of a concerted action of many weak chemical interactions of different types between the protein and its DNA target, including nonspecific electrostatic interactions, hydrogen bonding and van der Waals contacts. The precise complementarity of shape between the two macromolecules facilitates specific chemical recognition to be established. The electrophoretic mobility shift assay (EMSA) and several variants of footprinting are simple electrophoretic methods developed to study protein–DNA interactions. Because the specificity is determined by the nucleic acid sequence, the same methods can be exploited for a wide range of proteins simply by changing the sequence of the nucleic acid. EMSA detects sequence‐specific DNA‐binding activity in a protein sample as a separate migrating band in a nondenaturating gel. A footprinting method provides more detailed information on the precise location of a bound protein along the DNA fragment through the removal of specific bands in a pattern of cleaved fragments separated by electrophoresis. Both methods are highly sensitive due to the use of radioactively labeled oligonucleotides and can be performed with protein samples of low purity. When combined these methods are capable of providing a picture of the protein–DNA complex with a great deal of molecular detail, surpassed only by the more demanding methods of crystallography and nuclear magnetic resonance (NMR).

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Protein–Oligonucleotide Interactions

Gabrielsen

Matre

Bergholtz

2000

Encyclopedia of Analytical Chemistry

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Electrophoretic Mobility Shift Assays for the Analysis of DNA-Protein Interactions

Gaudreault

Gingras

Lessard

et al. 2009

Methods in Molecular Biology™

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Scanning for Transcription Factor Binding by a Variant EMSA

Gurevich

Zhang

Aneskievich

2009

Methods in Molecular Biology

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Detection of in vitro protein-DNA interaction is one of many investigational analyses for transcription factor regulation of gene promoters. The electrophoretic mobility shift assay (EMSA) has proven widely popular in this respect by integrating individual techniques (protein isolation, nucleic acid radiolabeling, and gel electrophoresis) into one protocol. However, relatively short DNA oligomers are often used which in many cases presupposes what one sequence out of a promoter of possibly thousands of base pairs is the candidate region interacting with a transcription factor. This can be an experimentally distressing situation when multiple putative binding sites of less than perfect consensus may be present making selection of any one or even a few potential sites uncertain or when one is seeking improved throughput as opposed to a one factor:one oligomer approach for in vitro testing of algorithm-predicted binding sites. We describe here our use and refinement of a variant EMSA that can employ multiple and relatively long (up to 1000 bp) probes of promoter sequence in one binding reaction for interaction with nuclear proteins in general and individual transcription factors in particular. We provide labeling and electrophoresis methods suitable for such probes and also highlight the mobility shift differences one can expect with the variant probe method.

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Improving Sensitivity of the Electrophoretic Mobility Shift Assay by Restricting Tissue Phosphatase Activities

Abstract: ABSTRACT

Cited by 4 publications

References 10 publications

Protein–Oligonucleotide Interactions

Protein–Oligonucleotide Interactions

Electrophoretic Mobility Shift Assays for the Analysis of DNA-Protein Interactions

Scanning for Transcription Factor Binding by a Variant EMSA

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