Analyzing the Nuclear Complexes of Notch Signaling by Electrophoretic Mobility Shift Assay
An electrophoretic mobility shift assay (EMSA) is a sensitive technique for detecting protein–DNA and protein–protein interactions in which complexes are separated by native (non-denaturing) gel electrophoresis. EMSAs can provide evidence for specific binding between components prepared from a wide range of sources, including not only highly purified proteins but also components of crude cellular extracts. EMSA experiments were critical in identifying the minimal protein requirements for assembly of transcriptionally active nuclear Notch complexes as well as the DNA sequence specificity of Notch transcription complexes. Here, we describe a radioactive EMSA protocol for detection of Notch transcription complexes.
Key wordsElectrophoretic mobility shift assay (EMSA) NICD CSL MAML1 RAMANK Protein–DNA interactions Bacterial protein expression and purification
This work was supported by NIH grants CA-092433 and CA-119070 (to S.C.B.).
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