Studies of Protein−DNA Interactions by Capillary Electrophoresis/Laser-Induced Fluorescence Polarization
Protein−DNA interactions were studied on the basis of capillary electrophoretic separation of bound from free fluorescent probe followed by on-line detection with laser-induced fluorescence polarization. Changes in electrophoretic mobility and fluorescence anisotropy upon complex formation were moni...
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| Veröffentlicht in: | Analytical chemistry (Washington) 2000-11, Vol.72 (22), p.5583-5589 |
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| creator | Wan, Qian-Hong Le, X. Chris |
| description | Protein−DNA interactions were studied on the basis of capillary electrophoretic separation of bound from free fluorescent probe followed by on-line detection with laser-induced fluorescence polarization. Changes in electrophoretic mobility and fluorescence anisotropy upon complex formation were monitored for the determination of binding affinity and stoichiometry. The method was applied to study the interactions of single-stranded DNA binding protein (SSB) with synthetic oligonucleotides and single-stranded DNA. Increases in fluorescence anisotropy and decreases in electrophoretic mobility upon their binding to SSB were observed for the fluorescently labeled 11-mer and 37-mer oligonucleotide probes. Fluorescence anisotropy and electrophoretic mobility were used to determine the binding constants of the SSB with the 11-mer (5 × 106 M-1) and the 37-mer (23 × 106 M-1). Alternatively, a fluorescently labeled SSB was used as a probe, and the formation of multiple protein−DNA complexes that differ in stoichiometry was observed. The results demonstrate the applicability of the method to study complex interactions between protein and DNA. |
| doi_str_mv | 10.1021/ac000318+ |
| format | Article |
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Chris</creator><creatorcontrib>Wan, Qian-Hong ; Le, X. Chris</creatorcontrib><description>Protein−DNA interactions were studied on the basis of capillary electrophoretic separation of bound from free fluorescent probe followed by on-line detection with laser-induced fluorescence polarization. Changes in electrophoretic mobility and fluorescence anisotropy upon complex formation were monitored for the determination of binding affinity and stoichiometry. The method was applied to study the interactions of single-stranded DNA binding protein (SSB) with synthetic oligonucleotides and single-stranded DNA. Increases in fluorescence anisotropy and decreases in electrophoretic mobility upon their binding to SSB were observed for the fluorescently labeled 11-mer and 37-mer oligonucleotide probes. Fluorescence anisotropy and electrophoretic mobility were used to determine the binding constants of the SSB with the 11-mer (5 × 106 M-1) and the 37-mer (23 × 106 M-1). 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Chris</creatorcontrib><title>Studies of Protein−DNA Interactions by Capillary Electrophoresis/Laser-Induced Fluorescence Polarization</title><title>Analytical chemistry (Washington)</title><addtitle>Anal. Chem</addtitle><description>Protein−DNA interactions were studied on the basis of capillary electrophoretic separation of bound from free fluorescent probe followed by on-line detection with laser-induced fluorescence polarization. Changes in electrophoretic mobility and fluorescence anisotropy upon complex formation were monitored for the determination of binding affinity and stoichiometry. The method was applied to study the interactions of single-stranded DNA binding protein (SSB) with synthetic oligonucleotides and single-stranded DNA. Increases in fluorescence anisotropy and decreases in electrophoretic mobility upon their binding to SSB were observed for the fluorescently labeled 11-mer and 37-mer oligonucleotide probes. Fluorescence anisotropy and electrophoretic mobility were used to determine the binding constants of the SSB with the 11-mer (5 × 106 M-1) and the 37-mer (23 × 106 M-1). Alternatively, a fluorescently labeled SSB was used as a probe, and the formation of multiple protein−DNA complexes that differ in stoichiometry was observed. The results demonstrate the applicability of the method to study complex interactions between protein and DNA.</description><subject>Base Sequence</subject><subject>Biological and medical sciences</subject><subject>Cellular biology</subject><subject>Deoxyribonucleic acid</subject><subject>DNA</subject><subject>DNA - chemistry</subject><subject>DNA Primers</subject><subject>DNA-Binding Proteins - chemistry</subject><subject>Electrophoresis, Capillary - methods</subject><subject>Fluorescence Polarization - methods</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Interactions. Associations</subject><subject>Intermolecular phenomena</subject><subject>Lasers</subject><subject>Molecular biophysics</subject><subject>Protein Binding</subject><subject>Proteins</subject><issn>0003-2700</issn><issn>1520-6882</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2000</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqF0dFqFDEUBuAgil2rF76ADCoiyNicZCbJXJZtqwuLLmzFy5DJnMGss5NtMgO2T9BrH9EnMcOuLeiFVwnJl59zcgh5DvQ9UAYnxlJKOah3D8gMSkZzoRR7SGbTac4kpUfkSYwbSgEoiMfkCKYN4-WMbNbD2DiMmW-zVfADuv7X7c-zT6fZoh8wGDs438esvs7mZue6zoTr7LxDOwS_--YDRhdPliZiyBd9M1pssotunM4t9hazlU8v3I2ZUp6SR63pIj47rMfky8X55fxjvvz8YTE_XeamqNSQKwTesLpAXqqKNQVAQ7G2ompbKAwKoUoGhtapzxqkLCshy7IUshKCq7oQ_Ji82efugr8aMQ5661I5qfYe_Ri1ZAWXBfD_wpReqBSd4Mu_4MaPoU9NaAZSCS4YTejtHtngYwzY6l1w2_RfGqiexqT_jCnRF4e8sd5icw8PY0ng1QGYaE3XBtNbF--cUlRySCrfKxcH_HF3a8J3LSSXpb5crfVqvj4rhPyqJ_96742N9x38U91v-auy4w</recordid><startdate>20001115</startdate><enddate>20001115</enddate><creator>Wan, Qian-Hong</creator><creator>Le, X. 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Chris</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-a498t-8e13d2b4e35892d411d0ebc69ff14ae668521a0b520b17759675556796638b463</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2000</creationdate><topic>Base Sequence</topic><topic>Biological and medical sciences</topic><topic>Cellular biology</topic><topic>Deoxyribonucleic acid</topic><topic>DNA</topic><topic>DNA - chemistry</topic><topic>DNA Primers</topic><topic>DNA-Binding Proteins - chemistry</topic><topic>Electrophoresis, Capillary - methods</topic><topic>Fluorescence Polarization - methods</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Interactions. 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Chris</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Studies of Protein−DNA Interactions by Capillary Electrophoresis/Laser-Induced Fluorescence Polarization</atitle><jtitle>Analytical chemistry (Washington)</jtitle><addtitle>Anal. Chem</addtitle><date>2000-11-15</date><risdate>2000</risdate><volume>72</volume><issue>22</issue><spage>5583</spage><epage>5589</epage><pages>5583-5589</pages><issn>0003-2700</issn><eissn>1520-6882</eissn><coden>ANCHAM</coden><abstract>Protein−DNA interactions were studied on the basis of capillary electrophoretic separation of bound from free fluorescent probe followed by on-line detection with laser-induced fluorescence polarization. Changes in electrophoretic mobility and fluorescence anisotropy upon complex formation were monitored for the determination of binding affinity and stoichiometry. The method was applied to study the interactions of single-stranded DNA binding protein (SSB) with synthetic oligonucleotides and single-stranded DNA. Increases in fluorescence anisotropy and decreases in electrophoretic mobility upon their binding to SSB were observed for the fluorescently labeled 11-mer and 37-mer oligonucleotide probes. Fluorescence anisotropy and electrophoretic mobility were used to determine the binding constants of the SSB with the 11-mer (5 × 106 M-1) and the 37-mer (23 × 106 M-1). Alternatively, a fluorescently labeled SSB was used as a probe, and the formation of multiple protein−DNA complexes that differ in stoichiometry was observed. The results demonstrate the applicability of the method to study complex interactions between protein and DNA.</abstract><cop>Washington, DC</cop><pub>American Chemical Society</pub><pmid>11101235</pmid><doi>10.1021/ac000318+</doi><tpages>7</tpages></addata></record> |
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| subjects | Base Sequence Biological and medical sciences Cellular biology Deoxyribonucleic acid DNA DNA - chemistry DNA Primers DNA-Binding Proteins - chemistry Electrophoresis, Capillary - methods Fluorescence Polarization - methods Fundamental and applied biological sciences. Psychology Interactions. Associations Intermolecular phenomena Lasers Molecular biophysics Protein Binding Proteins |
| title | Studies of Protein−DNA Interactions by Capillary Electrophoresis/Laser-Induced Fluorescence Polarization |
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