hFip1 (100 μM; residues 159–200 or 137-234) mutated to only have one cysteine that was outside of the CPSF30 binding region (S159C/C189S or S159C/C189S/C216S) was mixed with 2 mM fluorescein-5-maleimide (Cayman Chemical Company) in a buffer consisting of 20 mM Tris (pH 7.5), 100 mM NaCl, and 1 mM TCEP. The reaction was allowed to proceed overnight at 4°C in darkness. The next day, unreacted maleimide was rendered inactive by the addition of 10 mM DTT. The fluorescein-labeled hFip1 was then purified using a Superose 12 10/300 column (GE Healthcare).
Fluorescein 5 maleimide
Manufactured by Cayman Chemical
Fluorescein-5-maleimide is a fluorescent labeling reagent used for the detection and quantification of sulfhydryl-containing compounds. It forms a covalent bond with free sulfhydryl groups, allowing for the visualization and analysis of proteins, peptides, and other biomolecules.
Automatically generated - may contain errors
2 protocols using fluorescein 5 maleimide
1
Fluorescent Labeling of hFip1 Protein
2
Bacterial Siderophore Characterization
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Ferrichrome and fluorescein-5-maleimide were purchased from Cayman Chemicals. Ferrioxamine B was purchased from Calbiochem. Pyoverdine and Pyoverdine-Fe3+ were purchased from Sigma. Arthrobactin was purchased from MolPort. Alexa Fluor 594 C5 maleimide was purchased from Fisher Scientific. Carbenicillin was purchased from AK Scientific. l -arabinose was purchased from BioShop. Glucose was purchased from Fisher Scientific. Stock solutions and powders were stored at −20°C. Lysogeny broth (LB) was purchased as a premixed powder from BioShop. 10:90 medium was prepared as previously described (13 (link), 14 (link)). l -arabinose was prepared as a 20% (wt/vol) stock solution in 10:90 and filter sterilized (0.2-μm pore size; Fisherbrand).
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