Easily reversible desthiobiotin binding to streptavidin, avidin, and other biotin-binding proteins: uses for protein labeling, detection, and isolation.
AuthorsHirsch JD, Eslamizar L, Filanoski BJ, Malekzadeh N, Haugland RP, Beechem JM, Haugland RP
JournalAnal Biochem
PubMed ID12419349
'The high-affinity binding of biotin to avidin, streptavidin, and related proteins has been exploited for decades. However, a disadvantage of the biotin/biotin-binding protein interaction is that it is essentially irreversible under physiological conditions. Desthiobiotin is a biotin analogue that binds less tightly to biotin-binding proteins and is easily displaced by ... More
'Many areas of research today are based on enzymatic assays most of which are still performed as enzyme-linked immunosorbent assays in microtiter plates. The demand for highly parallel screening of thousands of samples eventually led to a miniaturization and automation of these assays. However, the final transfer of enzymatic assays ... More
Detection of cytochrome C in a single cell using an optical nanobiosensor.
AuthorsSong JM, Kasili PM, Griffin GD, Vo-Dinh T
JournalAnal Chem
PubMed ID15117202
'In this work, the intracellular measurement of cytochrome c using an optical nanobiosensor is demonstrated. The nanobiosensor is a unique fiberoptics-based tool which allows the minimally invasive analysis of intracellular components. Cytochrome c is a very important protein to the process which produces cellular energy. In addition, cytochrome c is ... More
A novel subfractionation approach for mitochondrial proteins: a three-dimensional mitochondrial proteome map.
AuthorsHanson BJ, Schulenberg B, Patton WF, Capaldi RA
JournalElectrophoresis
PubMed ID11332763
As mitochondria play critical roles in both cell life and cell death, there is great interest in obtaining a human mitochondrial proteome map. Such a map could potentially be useful in diagnosing diseases, identifying targets for drug therapy, and in screening for unwanted drug side effects. In this paper, we ... More
Transcript quantification based on chemical labeling of RNA associated with fluorescent detection.
AuthorsFontaine L, Even S, Soucaille P, Lindley ND, Cocaign-Bousquet M
JournalAnal Biochem
PubMed ID11700979
A general method for RNA measurement, based on chemical labeling of RNA with digoxigenin (without retrotranscription), has been established. Labeled RNA is hybridized with nylon membranes containing spot blots of PCR-amplified gene fragments and the fluorescence detection is mediated via specific anti-digoxigenin antibody coupled to alkaline phosphatase. The method was ... More
Activation of membrane-associated procaspase-3 is regulated by Bcl-2.
AuthorsKrebs JF, Armstrong RC, Srinivasan A, Aja T, Wong AM, Aboy A, Sayers R, Pham B, Vu T, Hoang K, Karanewsky DS, Leist C, Schmitz A, Wu JC, Tomaselli KJ, Fritz LC
JournalJ Cell Biol
PubMed ID10085291
The mechanism by which membrane-bound Bcl-2 inhibits the activation of cytoplasmic procaspases is unknown. Here we characterize an intracellular, membrane-associated form of procaspase-3 whose activation is controlled by Bcl-2. Heavy membranes isolated from control cells contained a spontaneously activatable caspase-3 zymogen. In contrast, in Bcl-2 overexpressing cells, although the caspase-3 ... More
Characterization of 9H-(1,3-dichlor-9, 9-dimethylacridin-2-ona-7-yl)-phosphate (DDAO) as substrate of PP-2A in a fluorimetric microplate assay for diarrhetic shellfish toxins (DSP).
AuthorsLeira F, Vieites JM, Vieytes MR, Botana LM
JournalToxicon
PubMed ID10858520
Specific inhibition of protein-phosphatases by diarrhetic shellfish toxins (DSP) of the okadaic acid group, has led to the development of a fluorescent enzyme inhibition assay for these toxins using protein-phosphatase 2A (PP-2A) and fluorogenic substrates of the enzyme. Two different substrates of PP-2A have been previously used in this microplate ... More
A miniature biochip system for detection of aerosolized Bacillus globigii spores.
AuthorsStratis-Cullum DN, Griffin GD, Mobley J, Vass AA, Vo-Dinh T
JournalAnal Chem
PubMed ID12553762
The feasibility of using a novel detection scheme for the analysis of biological warfare agents is demonstrated using Bacillus globigii spores, a surrogate species for Bacillus anthracis. In this paper, a sensitive and selective enzyme-linked immunosorbent assay using a novel fluorogenic alkaline phosphatase substrate (dimethylacridinone phosphate) is combined with a ... More
Single Molecule Detection Technologies in Miniaturized High Throughput Screening: Fluorescence Correlation Spectroscopy.
AuthorsMoore KJ, Turconi S, Ashman S, Ruediger M, Haupts U, Emerick V, Pope AJ
JournalJ Biomol Screen
PubMed ID10838431
Fluorescence assay technologies used for miniaturized high throughput screening are broadly divided into two classes. Macroscopic fluorescence techniques (encompassing conventional fluorescence intensity, anisotropy [also often referred to as fluorescence polarization] and energy transfer) monitor the assay volume- and time-averaged fluorescence output from the ensemble of emitting fluorophores. In contrast, single-molecule ... More
Mapping glycosylation changes related to cancer using the Multiplexed Proteomics technology: a protein differential display approach.
AuthorsSchulenberg B, Beechem JM, Patton WF
JournalJ Chromatogr B Analyt Technol Biomed Life Sci
PubMed ID12880860
The metastatic spread of tumor cells in malignant progression is known to be a major cause of cancer mortality. Protein glycosylation is increasingly being recognized as one of the most prominent biochemical alterations associated with malignant transformation and tumorigenesis. The Multiplexed Proteomics (MP) approach is a new technology that permits ... More
Simultaneous trichromatic fluorescence detection of proteins on Western blots using an amine-reactive dye in combination with alkaline phosphatase- and horseradish peroxidase-antibody conjugates.
AuthorsMartin K, Hart C, Liu J, Leung WY, Patton WF
JournalProteomics
PubMed ID12872222
Three-color fluorescence detection methods are described based upon covalently coupling the dye 2-methoxy-2,4-diphenyl-2(2H)-furanone (MDPF) to proteins immobilized on poly(vinylidene difluoride) (PVDF) membranes, followed by detection of target proteins using alkaline-phosphatase-conjugated reporter molecules in combination with the fluorogenic substrate 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) phosphate (DDAO-phosphate) as well as horseradish peroxidase-conjugated reporter molecules in combination ... More