EMSA
EMSA is a technique used to detect protein-nucleic acid interactions. Using specific and nonspecific probes designed for the experiment, when nucleic acid probes are incubated with protein samples, the proteins that can bind to the nucleic acid probes form protein-probe complexes. These complexes have a larger molecular weight and therefore migrate more slowly during PAGE electrophoresis, while the probes that do not bind to proteins migrate faster.
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Principle of EMSA
After incubation, PAGE electrophoresis, and membrane transfer, a band will form at the front of the membrane, indicating that a protein has interacted with the target probe.
Basic principle diagram of EMSA
Service process
Sample preparation — Probe labeling — Formation of protein-probe complexes — Preparation of EMSA gel — Electrophoresis — Membrane transfer — Detection — Submission of detailed experimental report
Delivery time
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Items |
Delivery time |
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Design and preparation of biotin-labeled probes |
1-2w |
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Protein extraction |
2w |
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EMSA gel electrophoresis mobility assay |
1-2w |
EMSA Advantages
- EMSA is easy to perform, simple, and does not require stringent experimental conditions, allowing for some flexibility in reaction conditions.
- Isotope-labeled probes have a very short half-life and are harmful to human health, while biotin-labeled probes are more stable, accurate, convenient, and have high affinity.
- The technique can detect a wide range of nucleic acid molecule sizes and structures (single-stranded, double-stranded, triple-stranded, quadruple-stranded, circular nucleic acids) and can provide the exact sequence position where proteins bind on the nucleic acid.
- Under appropriate conditions, the assay can detect the binding of different proteins to different nucleic acid molecules in a single reaction system by observing the formation of different complexes.
- EMSA can detect a wide range of protein sizes, from oligopeptides to transcription factor complexes, and can be performed with either purified proteins or cell extracts.
EMSA Disadvantages
- EMSA cannot account for the chemical equilibrium during electrophoresis; rapid dissociation occurring during electrophoresis may affect complex detection.
- The formation of complexes in EMSA is influenced by many factors and does not directly reflect molecular size or allow for the identification of various proteins in the complex.
- EMSA is largely a qualitative assay, meaning it can detect whether a protein-DNA complex forms but cannot finely measure the upregulation or downregulation of the binding reaction.
- EMSA does not take into account factors affecting protein-DNA binding in the in vivo environment, such as the influence of chromatin structure on complex formation. Thus, even if EMSA results indicate protein-DNA binding, additional conditions might be required to complete the binding reaction in vivo.
Therefore, EMSA is suitable for preliminary studies of protein-nucleic acid interactions.
