| Chapter title |
Shift-Western Blotting: Separate Analysis of Protein and DNA from Protein–DNA Complexes
|
|---|---|
| Chapter number | 36 |
| Book title |
Western Blotting
|
| Published in |
Methods in molecular biology, January 2015
|
| DOI | 10.1007/978-1-4939-2694-7_36 |
| Pubmed ID | |
| Book ISBNs |
978-1-4939-2693-0, 978-1-4939-2694-7
|
| Authors |
Matthias Harbers, Harbers, Matthias |
| Editors |
Biji T. Kurien, R. Hal Scofield |
| Abstract |
The electrophoretic mobility shift assay (EMSA) is the most frequently used experiment for studying protein-DNA interactions and to identify DNA-binding proteins. Protein-DNA complexes formed during EMSA experiments can be further analyzed by shift-western blotting, where the protein and DNA components contained in a polyacrylamide gel are transferred to stacked membranes: First a nitrocellulose membrane retains the proteins while double-stranded DNA passes through the nitrocellulose membrane and binds only to a charged membrane placed below. Immobilized proteins can then be stained with specific antibodies while the DNA can be detected by a radioactive label or a nonradioactive detection system. Shift-western blotting can overcome many limitations of supershift experiments and allows for the analysis of complex protein-DNA complexes containing multiple protein factors. Moreover, proteins and/or DNA may be recovered from membranes after the blotting step for further analysis by other means. |
Mendeley readers
Geographical breakdown
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|---|---|---|
| Unknown | 20 | 100% |
Demographic breakdown
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|---|---|---|
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| Student > Ph. D. Student | 4 | 20% |
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| Researcher | 2 | 10% |
| Other | 1 | 5% |
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| Unknown | 5 | 25% |
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|---|---|---|
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| Unknown | 5 | 25% |