'A fluorescent lactotransferrin probe was prepared by coupling 5-(([2-(carbhydrazino)methyl]-thio)acetyl)amino fluorescein to aldehyde groups that were produced by a mild periodic-acid oxidation of the glycan moieties of lactotransferrin. In this manner, the receptor-binding site of the lactotransferrin remains active in contrast to the binding site of the lactotransferrin derivatized with fluorescein ... More
Fluorescence-detected assembly of the signal recognition particle: binding of the two SRP protein heterodimers to SRP RNA is noncooperative.
AuthorsJaniak F, Walter P, Johnson AE
JournalBiochemistry
PubMed ID1377027
'Protein-RNA and protein-protein interactions involved in the assembly of the signal recognition particle (SRP) were examined using fluorescence spectroscopy. Fluorescein was covalently attached to the 3'-terminal ribose of SRP RNA following periodate oxidation, and the resulting SRP RNA-Fl was reconstituted into a fluorescent SRP species that was functional in promoting ... More
Reflection interference contrast microscopy combined with scanning force microscopy verifies the nature of protein-ligand interaction force measurements.
AuthorsStuart JK, Hlady V
JournalBiophys J
PubMed ID9876163
'The integration of a stand-alone scanning force microscope (SFM) scanner with a reflection interference contrast microscope (RICM) makes it possible to measure directly the separation distance between the SFM probe and the sample surface. The SFM-RICM combination, when applied to the force measurements between ligand-derivatized SFM probe and a protein ... More
Addition of the keto functional group to the genetic code of Escherichia coli.
AuthorsWang L, Zhang Z, Brock A, Schultz PG
JournalProc Natl Acad Sci U S A
PubMed ID12518054
Although the keto group is the most versatile of the functional groups in organic chemistry, it is absent in the genetically encoded amino acids. To overcome this natural limitation on protein biosynthesis, we have evolved an orthogonal tRNA-synthetase pair that makes possible the efficient incorporation of a keto amino acid, ... More
Site-specific labeling of cell surface proteins with biophysical probes using biotin ligase.
AuthorsChen I, Howarth M, Lin W, Ting AY
JournalNat Methods
PubMed ID15782206
We report a highly specific, robust and rapid new method for labeling cell surface proteins with biophysical probes. The method uses the Escherichia coli enzyme biotin ligase (BirA), which sequence-specifically ligates biotin to a 15-amino-acid acceptor peptide (AP). We report that BirA also accepts a ketone isostere of biotin as ... More
Synthesis and characterization of conjugates formed between periodate-oxidized ribonucleotides and amine-containing fluorophores.
AuthorsHileman RE, Parkhurst KM, Gupta NK, Parkhurst LJ
JournalBioconjug Chem
PubMed ID7849074
The synthesis and purification of new fluorescently labeled derivatives of GDP and ATP are described. The fluorescent groups are coupled initially through amine-containing linker arms to periodate-oxidized nucleotides. Reduction of the initial product yields primarily a six-membered morpholine-like ring. Fluorescein-labeled GDP, rhodamine-labeled GDP, and fluorescein-labeled ATP were characterized by absorbance ... More
Fluorescence labeling of bacteria for studies of intracellular pathogenesis.
AuthorsDrevets DA, Elliott AM
JournalJ Immunol Methods
PubMed ID7490459
Interactions between intracellular bacterial pathogens and their eukaryotic cellular hosts or targets are often studied with fluorescence-based techniques such as fluorescence microscopy and flow cytometry. We tested whether the intracellular bacterial pathogens L. monocytogenes, M. avium, M. tuberculosis, and S. typhimurium could be labeled by growth in broth containing the ... More
Internalization of human lactoferrin by the Jurkat human lymphoblastic T-cell line.
AuthorsBi BY, Liu JL, Legrand D, Roche AC, Capron M, Spik G, Mazurier J
JournalEur J Cell Biol
PubMed ID8900493
Binding of either iron-saturated or iron-free lactoferrin to the Jurkat human lymphoblastic T-cell line was saturable with a dissociation constant Kd of 40 nM. The total number of binding sites was estimated to be approximately 300,000. Non-specific binding did not exceed 30% of the total binding. Removal of the 4 ... More
Fluorescent labelling of abasic sites: a novel methodology to detect closely-spaced damage sites in DNA.
AuthorsMakrigiorgos GM, Chakrabarti S, Mahmood A
JournalInt J Radiat Biol
PubMed ID9687979
PURPOSE: To test the ability of a new fluorescent reagent to label abasic sites in DNA and to use fluorescent energy transfer as a measure of closely-spaced abasic sites in DNA. MATERIALS AND METHODS: A fluorescein-conjugated hydroxylamine derivative (FARP, 5-(((2-(carbohydrazino)-methyl)thio)acetyl)aminofluorescein, aminooxyacetyl hydrazide) that reacts covalently with open chain aldehydes in ... More