Source: Singh, R. Exploring Sequence Space to Identify Binding Sites for Regulatory RNA-Binding Proteins. J. Vis. Exp. ( (2019).
In this video, we describe the systematic evolution of ligands by the exponential enrichment (SELEX) method to identify specific RNA-binding sequences for a target protein of interest. The protein is incubated with a large pool of randomized RNA sequences, and the protein-binding RNA sequences are isolated, PCR-amplified, and sequenced to identify their protein-binding sites.
1. Protein binding reaction and separation of bound RNA
2. Reverse transcription and PCR amplification
3. Transcription and protein binding
The authors have nothing to disclose.
Gel Electrophoresis equipment | Standard | Standard | |
Nitrocellulose | Millipore | HAWP | |
Nitrocellulose | Schleicher & Schuell | PROTRAN | |
Polyacrylamide gel solutions | Standard | Standard | |
Proteinase K | NEB | P8107S | |
Recombinant PTB | Laboratory Preparation | Not applicable | |
Reverse Transcriptase | NEB | M0277S | |
Vacuum manifold | Fisher Scientific | XX1002500 | Millipore 25mm Glass Microanalysis Vacuum Filter |
Vacuum manifold | Millipore | XX2702552 | 1225 Sampling Vacuum Manifold |
X-ray films | Standard | Standard | |
Glass Plates | Standard | Standard |