TY - CHAP
T1 - Standard in vitro assays for protein-nucleic acid interactions - Gel shift assays for RNA and DNA binding
AU - Mitchell, Sarah F.
AU - Lorsch, Jon R.
PY - 2014
Y1 - 2014
N2 - The characterization of protein-nucleic acid interactions is necessary for the study of a wide variety of biological processes. One straightforward and widely used approach to this problem is the electrophoretic mobility shift assay (EMSA), in which the binding of a nucleic acid to one or more proteins changes its mobility through a nondenaturing gel matrix. Usually, the mobility of the nucleic acid is reduced, but examples of increased mobility do exist. This type of assay can be used to investigate the affinity of the interaction between the protein and nucleic acid, the specificity of the interaction, the minimal binding site, and the kinetics of the interaction. One particular advantage of EMSA is the ability to analyze multiple proteins, or protein complexes, binding to nucleic acids. This assay is relatively quick and easy and utilizes equipment available in most laboratories; however, there are many variables that can only be determined empirically; therefore, optimization is necessary and can be highly dependent upon the system. The protocol described here is for the poly(A)-binding protein (PABP) binding to an unstructured RNA probe of 43 bases. While this may be a useful protocol for some additional assays, it is recommended that both reaction conditions and gel running conditions be tailored to the individual interaction to be probed.
AB - The characterization of protein-nucleic acid interactions is necessary for the study of a wide variety of biological processes. One straightforward and widely used approach to this problem is the electrophoretic mobility shift assay (EMSA), in which the binding of a nucleic acid to one or more proteins changes its mobility through a nondenaturing gel matrix. Usually, the mobility of the nucleic acid is reduced, but examples of increased mobility do exist. This type of assay can be used to investigate the affinity of the interaction between the protein and nucleic acid, the specificity of the interaction, the minimal binding site, and the kinetics of the interaction. One particular advantage of EMSA is the ability to analyze multiple proteins, or protein complexes, binding to nucleic acids. This assay is relatively quick and easy and utilizes equipment available in most laboratories; however, there are many variables that can only be determined empirically; therefore, optimization is necessary and can be highly dependent upon the system. The protocol described here is for the poly(A)-binding protein (PABP) binding to an unstructured RNA probe of 43 bases. While this may be a useful protocol for some additional assays, it is recommended that both reaction conditions and gel running conditions be tailored to the individual interaction to be probed.
KW - Coomassie blue staining
KW - Electrophoretic mobility shift assay (EMSA)
KW - Gel shift assays
KW - In vitro assays
KW - Poly(A)-binding protein (PABP)
KW - Polyacrylamide gel preparation
KW - Protein-nucleic acid interactions
KW - RNA and DNA binding
UR - http://www.scopus.com/inward/record.url?scp=84896945913&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84896945913&partnerID=8YFLogxK
U2 - 10.1016/B978-0-12-420119-4.00015-X
DO - 10.1016/B978-0-12-420119-4.00015-X
M3 - Chapter
C2 - 24674072
AN - SCOPUS:84896945913
SN - 9780124201194
T3 - Methods in Enzymology
SP - 179
EP - 196
BT - Laboratory Methods in Enzymology
PB - Academic Press Inc.
ER -