Electrophoretic mobility shift assay for the detection of specific DNA-protein complex in nuclear extracts from the cultured cells and frozen autopsy human brain tissue

@article{citeulike:2776058, abstract = {The electrophoretic mobility shift assay (EMSA) is generally used to study the interaction of transcription factors to specific DNA sequences. The preparation of high quality nuclear extracts is an important step before performing the assay. Here we describe a rapid method for the isolation of good-quality DNA-binding proteins from cultured cell lines and autopsy tissue samples from the human brain. The [`]rapid method' (RM) utilizes the low salt/detergent lysis steps followed by high salt extraction of nuclei. To test and compare the activity of nuclear extracts prepared by the standard and [`]rapid' methods for its ability to form the specific DNA-protein complex, EMSA was carried out with three different oligonucleotide probes: AP1, NF-[kappa]B and URE. A comparative study indicates that the capacity to form the specific DNA-protein complex with these oligonucleotide probes by standard and RM nuclear extracts was very similar. Each nuclear extract formed the corresponding DNA-protein complex, the specificity of which was checked by the competition experiment. In some cases unspecific bands were observed and which were present in nuclear extracts from both preparations. Thus the simplicity of the [`]rapid method' permits the preparation of nuclear extracts from several cell lines and tissue samples at the same time at much shorter time than the [`]standard' method without compromising the DNA-binding activity. The RM can be applied to determine the cell type or tissue specificity of transcription factors in an efficient, economical and consistent manner.}, author = {Lahiri, Debomoy K. and Ge, Yuan-Wen }, citeulike-article-id = {2776058}, doi = {10.1016/S1385-299X(00)00021-0}, journal = {Brain Research Protocols}, month = {July}, number = {3}, pages = {257--265}, posted-at = {2008-05-09 15:56:06}, priority = {2}, title = {Electrophoretic mobility shift assay for the detection of specific DNA-protein complex in nuclear extracts from the cultured cells and frozen autopsy human brain tissue}, url = {http://dx.doi.org/10.1016/S1385-299X(00)00021-0}, volume = {5}, year = {2000} }

TY - JOUR ID - citeulike:2776058 L3 - citeulike-article-id:2776058 TI - Electrophoretic mobility shift assay for the detection of specific DNA-protein complex in nuclear extracts from the cultured cells and frozen autopsy human brain tissue JF - Brain Research Protocols VL - 5 IS - 3 SP - 257 EP - 265 N2 - The electrophoretic mobility shift assay (EMSA) is generally used to study the interaction of transcription factors to specific DNA sequences. The preparation of high quality nuclear extracts is an important step before performing the assay. Here we describe a rapid method for the isolation of good-quality DNA-binding proteins from cultured cell lines and autopsy tissue samples from the human brain. The [`]rapid method' (RM) utilizes the low salt/detergent lysis steps followed by high salt extraction of nuclei. To test and compare the activity of nuclear extracts prepared by the standard and [`]rapid' methods for its ability to form the specific DNA-protein complex, EMSA was carried out with three different oligonucleotide probes: AP1, NF-[kappa]B and URE. A comparative study indicates that the capacity to form the specific DNA-protein complex with these oligonucleotide probes by standard and RM nuclear extracts was very similar. Each nuclear extract formed the corresponding DNA-protein complex, the specificity of which was checked by the competition experiment. In some cases unspecific bands were observed and which were present in nuclear extracts from both preparations. Thus the simplicity of the [`]rapid method' permits the preparation of nuclear extracts from several cell lines and tissue samples at the same time at much shorter time than the [`]standard' method without compromising the DNA-binding activity. The RM can be applied to determine the cell type or tissue specificity of transcription factors in an efficient, economical and consistent manner. AU - Lahiri, Debomoy AU - Ge, Yuan-Wen PY - 2000/07// UR - http://dx.doi.org/10.1016/S1385-299X(00)00021-0 ER -