Sortase-mediated fluorescent labeling of CRISPR complexes
| Autor: | Samuel D. Dahlhauser, Yibei Xiao, Fatema A. Saifuddin, Erik T. Hernandez, Ilya J. Finkelstein, Jeffrey M. Schaub, Kaylee E. Dillard, Eric V. Anslyn, Ailong Ke, Maxwell W. Brown |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2018 |
| Předmět: |
Models
Molecular 030303 biophysics CRISPR-Associated Proteins Peptide Epitope Article 03 medical and health sciences chemistry.chemical_compound Sortase Escherichia coli CRISPR Clustered Regularly Interspaced Short Palindromic Repeats Fluorescent Dyes chemistry.chemical_classification 0303 health sciences Staining and Labeling Chemistry Escherichia coli Proteins Optical Imaging Fluorescence Amino acid Cysteine Endopeptidases Biochemistry Target protein CRISPR-Cas Systems DNA |
| Popis: | Fluorescent labeling of proteins is a critical requirement for single-molecule imaging studies. Many protein labeling strategies require harsh conditions or large epitopes that can inactivate the target protein, either by decreasing the protein's enzymatic activity or by blocking protein-protein interactions. Here, we provide a detailed protocol to efficiently label CRISPR-Cas complexes with a small fluorescent peptide via sortase-mediated transpeptidation. The sortase tag consists of just a few amino acids that are specifically recognized at either the N- or the C-terminus, making this strategy advantageous when the protein is part of a larger complex. Sortase is active at high ionic strength, 4°C, and with a broad range of organic fluorophores. We discuss the design, optimization, and single-molecule fluorescent imaging of CRISPR-Cas complexes on DNA curtains. Sortase-mediated transpeptidation is a versatile addition to the protein labeling toolkit. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|