Sensitive fluorescence-based thermodynamic and kinetic measurements of DNA hybridization in solution.
AuthorsMorrison LE,Stols LM
JournalBiochemistry
PubMed ID8457571
Kinetic and thermodynamic constants associated with DNA hybridization were determined in solution using fluorescence measurements and complementary fluorophore-labeled oligomers. One oligomer was labeled with a 5'-terminal fluorescein, and the other was labeled with a 3'-terminal rhodamine. The juxtaposition of the two labels in double-stranded complexes results in a strong quenching ... More
SH2-B is required for growth hormone-induced actin reorganization.
AuthorsHerrington J, Diakonova M, Rui L, Gunter DR, Carter-Su C
JournalJ Biol Chem
PubMed ID10777618
'The Src homology-2 (SH2) domain-containing protein SH2-Bbeta is a substrate of the growth hormone (GH) receptor-associated tyrosine kinase JAK2. Here we tested whether SH2-Bbeta is involved in GH regulation of the actin cytoskeleton. Based on cell fractionation and confocal microscopy, we find SH2-Bbeta present at the plasma membrane and in ... More
Alexa dyes, a series of new fluorescent dyes that yield exceptionally bright, photostable conjugates.
AuthorsPanchuk-Voloshina N, Haugland RP, Bishop-Stewart J, Bhalgat MK, Millard PJ, Mao F, Leung WY, Haugland RP
JournalJ Histochem Cytochem
PubMed ID10449539
'Alexa 350, Alexa 430, Alexa 488, Alexa 532, Alexa 546, Alexa 568, and Alexa 594 dyes are a new series of fluorescent dyes with emission/excitation spectra similar to those of AMCA, Lucifer Yellow, fluorescein, rhodamine 6G, tetramethylrhodamine or Cy3, lissamine rhodamine B, and Texas Red, respectively (the numbers in the ... More
Kinetics of antigenic peptide binding to the class II major histocompatibility molecule I-Ad.
AuthorsTampé R, McConnell HM
JournalProc Natl Acad Sci U S A
PubMed ID2052549
'Using high-performance size-exclusion chromatography and fluorescence spectroscopy, we investigated the kinetics of fluorescent peptide reactions with detergent-solubilized I-Ad, a class II molecule of the mouse major histocompatibility complex. At pH 7.0 and 37 degrees C the half-time for the binding of a fluorescein-labeled synthetic peptide representing ovalbumin amino acids 323-339 ... More
Co-expression of chemotactic ligand receptors on human peripheral blood monocytes.
AuthorsOhura K, Katona IM, Wahl LM, Chenoweth DE, Wahl SM
JournalJ Immunol
PubMed ID2435802
'Directed migration of monocytes is dependent upon interaction of cell surface receptors and specific chemotactic ligands. To determine whether circulating human monocytes express multiple chemotactic ligand receptors or whether subpopulations of monocytes exist with a single receptor specificity, nonoverlapping fluorescent probes for two chemotactic ligands, N-formyl methionyl leucyl phenylalanine (FMLP) ... More
Specific associations of fluorescent beta-2-microglobulin with cell surfaces. The affinity of different H-2 and HLA antigens for beta-2-microglobulin.
AuthorsHochman JH, Shimizu Y, DeMars R, Edidin M
JournalJ Immunol
PubMed ID2450918
'We prepared single-labeled FITC derivatives of beta-2-microglobulin (b2m) and examined their interactions with class I MHC Ag H chains on living cells. Human b2m was reacted with FITC under mild conditions and separated by hydroxylapatite chromatography into three peaks containing single labeled derivatives of b2m peaks A, B, and C, ... More
Flow cytometric quantitation of oxidative product formation by polymorphonuclear leukocytes during phagocytosis.
AuthorsSzejda P, Parce JW, Seeds MS, Bass DA
JournalJ Immunol
PubMed ID6491287
'Stimulation of the oxidative metabolic burst of human polymorphonuclear leukocytes (PMNL) may occur by an all-or-none trigger mechanism or by a graded response to increasing stimulation of an individual cell. If the proposed all-or-none mechanism occurred during phagocytosis, a PMNL would expend all of its metabolic potential at once, yet ... More
The receptor recycling pathway contains two distinct populations of early endosomes with different sorting functions.
AuthorsSheff DR, Daro EA, Hull M, Mellman I
JournalJ Cell Biol
PubMed ID10189373
'Receptor recycling involves two endosome populations, peripheral early endosomes and perinuclear recycling endosomes. In polarized epithelial cells, either or both populations must be able to sort apical from basolateral proteins, returning each to its appropriate plasma membrane domain. However, neither the roles of early versus recycling endosomes in polarity nor ... More
Quantitative analysis of TIP47-receptor cytoplasmic domain interactions: implications for endosome-to-trans Golgi network trafficking.
AuthorsKrise JP, Sincock PM, Orsel JG, Pfeffer SR
JournalJ Biol Chem
PubMed ID10829017
'TIP47 (tail-interacting protein of 47 kDa) binds to the cytoplasmic domains of the cation-independent and cation-dependent mannose 6-phosphate receptors and is required for their transport from late endosomes to the trans Golgi network in vitro and in vivo. We report here a quantitative analysis of the interaction of recombinant TIP47 ... More
Quantitative analysis of Fc gamma receptors on murine spleen cell populations by using dual parameter flow cytometry.
AuthorsTitus JA, Finkelman FD, Stephany DA, Jones JF, Segal DM
JournalJ Immunol
PubMed ID6234350
'The expression of Fc gamma R on subsets of mouse spleen cells was examined by dual parameter flow microfluorometry. B cells were detected by labeling them with antibodies against sIgM, sIgD, sIgG, or I-A; essentially all B cells expressed Fc gamma R. The number of Fc gamma R per cell ... More
Cell death-associated translocation of plasma membrane components induced by CTL.
AuthorsKawasaki Y, Saito T, Shirota-Someya Y, Ikegami Y, Komano H, Lee MH, Froelich CJ, Shinohara N, Takayama H
JournalJ Immunol
PubMed ID10779768
'In the very early stages of target cell apoptosis induced by CTL, we found that fluorescence of labeling probes of the target plasma membrane, such as N-(3-triethylammoniumpropyl)-4-(p-dibutylaminostyryl)pyridin ium dibromide (FM1-43), was translocated into intracellular membrane structures including nuclear envelope and mitochondria. This translocation was associated with the execution of CTL-mediated ... More
A comparison of non-radioisotopic hybridization assay methods using fluorescent, chemiluminescent and enzyme labeled synthetic oligodeoxyribonucleotide probes.
AuthorsUrdea MS, Warner BD, Running JA, Stempien M, Clyne J, Horn T
JournalNucleic Acids Res
PubMed ID3387214
'N4-[N-(6-trifluoroacetylamidocaproyl)-2-aminoethyl]-5''-O-dimethoxy trityl -5-methyl-2''-deoxycytidine-3''-N,N-diisopropyl-methylphosphoramidite++ + has been synthesized. This N4-alkylamino deoxycytidine derivative has been incorporated into oligonucleotide probes during chemical DNA synthesis. Subsequent to deprotection and purification, fluorescent (fluorescein, Texas Red and rhodamine), chemiluminescent (isoluminol), and enzyme (horseradish peroxidase, alkaline phosphatase) labels have been specifically incorporated. Detection limits of the labels ... More
Flow cytometric quantitation of human opsonin-dependent phagocytosis and oxidative burst responses to meningococcal antigens.
AuthorsLehmann AK, Halstensen A, Bassøe CF
JournalCytometry
PubMed ID9845434
'A one-step flow cytometric (FCM) assay has been developed to quantify both opsonin- and antigen-dependent phagocytosis and intraphagocyte oxidative burst responses. Meningococcal outer membrane structures (OMV) were adsorbed to fluorescent polystyrene beads, opsonized with serum, and exposed to leukocytes. FCM parameters of phagocytosis were evaluated in combinations with oxidative burst ... More
Distribution and function of nitric oxide-containing nerves in canine corpus cavernosum and spongiosum.
AuthorsHedlund P, Larsson B, Alm P, Andersson KE
JournalActa Physiol Scand
PubMed ID8719264
'Specimens of penile erectile tissue from the corpus cavernosum (CC) and corpus spongiosum (CS) of beagle dogs were investigated with reference to morphological and functional aspects of the nitric oxide (NO) system. Using immunohistochemistry, the smooth muscle bundles of the CC were found to contain a rich innervation, as seen ... More
Characterization of cellular optoporation with distance.
'We have developed and characterized cellular optoporation with visible wavelengths of light using standard uncoated glass cover slips as the absorptive media. A frequency-doubled Nd:YAG laser pulse was focused at the interface of the glass surface and aqueous buffer, creating a stress wave and transiently permeabilizing nearby cells. Following optoporation ... More
Pyrularia thionin binding to and the role of tryptophan-8 in the enhancement of phosphatidylserine domains in erythrocyte membranes.
AuthorsWang F, Naisbitt GH, Vernon LP, Glaser M
JournalBiochemistry
PubMed ID8241114
'Pyrularia thionin is a small, strongly basic peptide which interacts readily with cellular and synthetic membranes. With cells it induces hemolysis, depolarizes the cellular membrane with an accompanying influx of Ca2+, and activates an endogenous phospholipase A2. Evidence points toward a binding site involving phosphatidylserine (PS). This study shows that ... More
Lipid-modified, cysteinyl-containing peptides of diverse structures are efficiently S-acylated at the plasma membrane of mammalian cells.
AuthorsSchroeder H, Leventis R, Shahinian S, Walton PA, Silvius JR
JournalJ Cell Biol
PubMed ID8707845
'A variety of cysteine-containing, lipid-modified peptides are found to be S-acylated by cultured mammalian cells. The acylation reaction is highly specific for cysteinyl over serinyl residues and for lipid-modified peptides over hydrophilic peptides. The S-acylation process appears by various criteria to be enzymatic and resembles the S-acylation of plasma membrane-associated ... More
Spectra and fluorescence lifetimes of lissamine rhodamine, tetramethylrhodamine isothiocyanate, texas red, and cyanine 3.18 fluorophores: influences of some environmental factors recorded with a confocal laser scanning microscope.
AuthorsBrismar H, Trepte O, Ulfhake B
JournalJ Histochem Cytochem
PubMed ID7608524
'We report on the spectra and fluorescence lifetimes of four commonly used fluorophores: lissamine rhodamine (LRSC); tetramethyl rhodamine isothiocyanate (TRITC); Texas Red; and cyanine 3.18 (Cy-3). Fluorescence lifetime recordings revealed that these spectrally overlapping fluorophores can be individually detected by their lifetimes, indicating that at least four fluorophores can be ... More
Analysis by confocal scanning laser microscopy imaging of the spatial distribution of intermediate filaments in foetal and adult rat liver cells.
AuthorsVassy J, Rigaut JP, Hill AM, Foucrier J
JournalJ Microsc
PubMed ID1688949
'Confocal scanning laser microscopy has been used to make three-dimensional observations of the spatial distribution of cytoskeleton intermediate filaments in rat liver hepatocytes, at various stages during foetal development and in the adult. Single and double immuno-labelling with fluorescein and Texas Red fluorescence have been used to study the intracellular ... More
Photophysical analysis of class I major histocompatibility complex protein assembly using a xanthene-derivatized beta2-microglobulin.
AuthorsGakamsky DM, Davis DM, Haas E, Strominger JL, Pecht I
JournalBiophys J
PubMed ID10049335
'Spectral changes and a sixfold increase in the emission intensity were observed in the fluorescence of a single xanthene probe (Texas red) attached to beta2m-microglobulin (beta2m) upon assembly of beta2m into a ternary complex with mouse H-2Kd heavy chain and influenza nuclear protein peptide. Dissociation of the labeled beta2m from ... More
Two-photon fluorescence coincidence analysis: rapid measurements of enzyme kinetics.
AuthorsHeinze KG, Rarbach M, Jahnz M, Schwille P
JournalBiophys J
PubMed ID12202390
'Dual-color fluorescence cross-correlation analysis is a powerful tool for probing interactions of different fluorescently labeled molecules in aqueous solution. The concept is the selective observation of coordinated spontaneous fluctuations in two separate detection channels that unambiguously reflect the existence of physical or chemical linkages among the different fluorescent species. It ... More
Hydrogel-based protein microchips: manufacturing, properties, and applications.
AuthorsRubina AY, Dementieva EI, Stomakhin AA, Darii EL, Pan'kov SV, Barsky VE, Ivanov SM, Konovalova EV, Mirzabekov AD
JournalBiotechniques
PubMed ID12765028
'Here a simple, reproducible, and versatile method is described for manufacturing protein and ligand chips. The photo-induced copolymerization of acrylamide-based gel monomers with different probes (oligonucleotides, DNA, proteins, and low-molecular ligands) modified by the introduction of methacrylic groups takes place in drops on a glass or silicone surface. All probes ... More
High-performance optical filters for fluorescence analysis.
AuthorsMarcus DA
JournalCell Motil Cytoskeleton
PubMed ID3180249
'Recent advances in thin film optical coating technology significantly improve the filters available for fluorescence spectroscopy. Bandpass and long- and shortpass filters with very sharply defined edges can provide from 10(-5) to 10(-6) blocking within 10-15 nm of the transmission region and are ideal for use as excitation and emission ... More
Fluorescent labeling of cell surfaces.
AuthorsEdidin M
JournalMethods Cell Biol
PubMed ID2464121
The synthesis and use of fluorescent oligonucleotides in DNA sequence analysis.
AuthorsSmith LM, Kaiser RJ, Sanders JZ, Hood LE
JournalMethods Enzymol
PubMed ID3431463
High-throughput SNP genotyping by allele-specific PCR with universal energy-transfer-labeled primers.
AuthorsMyakishev MV, Khripin Y, Hu S, Hamer DH
JournalGenome Res
PubMed ID11156625
'We have developed a new method for high-throughput genotyping of single nucleotide polymorphisms (SNPs). The technique involves PCR amplification of genomic DNA with two tailed allele-specific primers that introduce priming sites for universal energy-transfer-labeled primers. The output of red and green light is conveniently scored using a fluorescence plate reader. ... More
Synaptic differentiation can be evoked by polymer microbeads that mimic localized pericellular proteolysis by removing proteins from adjacent surfaces.
AuthorsAnderson MJ, Champaneria S, Swenarchuk LE
JournalDev Biol
PubMed ID1916019
'Synaptic differentiation is normally "induced" by regulatory signals that are exchanged only at close contacts between neurites and their predetermined target cells. These signals can, however, be mimicked by contact of either cell with some kinds of polymer microbeads. To find what bead action is responsible for this mimicry, we ... More
High-affinity fluorescent peptide binding to I-Ad in lipid membranes.
AuthorsWatts TH, McConnell HM
JournalProc Natl Acad Sci U S A
PubMed ID2948183
'Membranes isolated from antigen-pulsed, antigen-presenting cells were extensively dialyzed and shown to retain the ability to present antigen to an I-Ad-restricted, antigen-specific T cell, 3DO-54.8. This ability to retain antigen on the membrane was duplicated in lysates of antigen-presenting cells as well as with pure I-A molecules in phospholipid vesicles. ... More
Quantification of antibodies to human growth hormone by high-performance protein G affinity chromatography with fluorescence detection.
AuthorsRiggin A, Regnier FE, Sportsman JR
JournalAnal Chem
PubMed ID2064010
'The technique of high-performance affinity chromatography (HPAC) is applied to the quantitative determination of antibodies to human growth hormone (hGH) in serum from patients. An affinity column consisting of covalently immobilized protein G on a rigid support is used to capture the antibodies. Texas Red labeled hGH (hGH-TR) is used ... More
Resolving leukocytes using axial light loss.
AuthorsStewart CC, Stewart SJ, Habbersett RC
JournalCytometry
PubMed ID2766889
'Axial light loss (ALL) is the measurement of the total light lost from the laser beam at 0 degrees when a particle passes through the beam. Used in combination with the monoclonal antibody CD45, ALL can effectively resolve lymphocytes, monocytes, granulocytes, and dead cells in viable or fixed preparations of ... More
Interactions of pulmonary surfactant protein A with phospholipid monolayers change with pH.
AuthorsRuano ML, Nag K, Casals C, Pérez-Gil J, Keough KM
JournalBiophys J
PubMed ID10465757
'The interaction of pulmonary surfactant protein A (SP-A) labeled with Texas Red (TR-SP-A) with monolayers containing zwitterionic and acidic phospholipids has been studied at pH 7.4 and 4.5 using epifluorescence microscopy. At pH 7.4, TR-SP-A expanded the pi-A isotherms of film of dipalmitoylphosphatidylcholine (DPPC). It interacted at high concentration at ... More
Dehydroascorbic acid prevents apoptosis induced by oxidized low-density lipoprotein in human monocyte-derived macrophages.
AuthorsAsmis R, Wintergerst ES
JournalEur J Biochem
PubMed ID9692913
'Human low-density lipoprotein (LDL) oxidized with Cu2+ or the radical generator 2,2''-azobis(2-methyl-propionamidine) hydrochloride (AAPH) induces apoptosis in mature human monocyte-derived macrophages as assessed by staining with fluorescein-isothiocyanate-labeled annexin V, by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling, and by staining of the 7A6 mitochondrial antigen. Oxidized LDL-induced apoptosis was dose and ... More
Fluorescence detection in automated DNA sequence analysis.
AuthorsSmith LM, Sanders JZ, Kaiser RJ, Hughes P, Dodd C, Connell CR, Heiner C, Kent SB, Hood LE
JournalNature
PubMed ID3713851
'We have developed a method for the partial automation of DNA sequence analysis. Fluorescence detection of the DNA fragments is accomplished by means of a fluorophore covalently attached to the oligonucleotide primer used in enzymatic DNA sequence analysis. A different coloured fluorophore is used for each of the reactions specific ... More
The use of fluorescent probes in immunochemistry.
AuthorsDavidson RS, Hilchenbach MM
JournalPhotochem Photobiol
PubMed ID2217553
'The limitations and advantages of particular dyes for labelling proteins and other biological materials are discussed. Methods available for conjugating dyes to proteins are outlined. Following a discussion of double labelling methods the use of photoactivatable fluorochromes and time resolved fluorescence methodologies are outlined. The reasons for the photoinstability of ... More
Three-color immunofluorescence histochemistry allowing triple labeling within a single section.
AuthorsStaines WA, Meister B, Melander T, Nagy JI, Hökfelt T
JournalJ Histochem Cytochem
PubMed ID2891745
'We describe a procedure for simultaneous immunohistochemical localization of three different neuropeptides, neurotransmitters, or neurotransmitter enzymes within one and the same tissue section and present a number of examples of its application within the brain and periphery. Primary antibodies from three different species were bound to three different neurochemical substances ... More
Immunohistochemical evidence for the coexistence of histidine decarboxylase-like and glutamate decarboxylase-like immunoreactivities in nerve cells of the magnocellular nucleus of the posterior hypothalamus of rats.
AuthorsTakeda N, Inagaki S, Shiosaka S, Taguchi Y, Oertel WH, Tohyama M, Watanabe T, Wada H
JournalProc Natl Acad Sci U S A
PubMed ID6594708
'Immunohistochemical staining of alternate consecutive sections revealed numerous histidine decarboxylase (L-histidine carboxy-lyase, EC 4.1.1.22)-like immunoreactive neurons that also contained glutamate decarboxylase (L-glutamate 1-carboxy-lyase, EC 4.1.1.15)-like immunoreactive structures in the tuberal magnocellular nucleus, the caudal magnocellular nucleus, and the postmammillary caudal magnocellular nucleus of the posterior hypothalamus of rats. Furthermore, in ... More
Determining nuclear structure using the fluorescence light microscope.
AuthorsPaddy MR
JournalMethods Cell Biol
PubMed ID9348504
Fluorescence-activated cell sorting: theory, experimental optimization, and applications in lymphoid cell biology.
AuthorsParks DR, Herzenberg LA
JournalMethods Enzymol
PubMed ID6396481
Multicolor laser scanning confocal immunofluorescence microscopy: practical application and limitations.
AuthorsBrelje TC, Wessendorf MW, Sorenson RL
JournalMethods Cell Biol
PubMed ID8246789
Preparation of a fluorescent analog: acetamidofluoresceinyl-labeled Dictyostelium discoideum alpha-actinin.
AuthorsSimon JR, Taylor DL
JournalMethods Enzymol
PubMed ID3029546
Homogeneous immunochemical technique for determination of human lactoferrin using excitation transfer and phase-resolved fluorometry.
AuthorsNithipatikom K, McGown LB
JournalAnal Chem
PubMed ID3551686
Protein labeling with fluorescent probes.
AuthorsHolmes KL, Lantz LM
JournalMethods Cell Biol
PubMed ID11060842
Fluorescent localization of contractile proteins in tissue culture cells.
AuthorsWang K, Feramisco JR, Ash JF
JournalMethods Enzymol
PubMed ID6750319
Detection of doubly stained fluorescent specimens using confocal microscopy.
AuthorsMossberg K, Ericsson M
JournalJ Microsc
PubMed ID1695246
Studies of doubly stained specimens were performed with a confocal scanning microscope. The instrument used provides the possibility of making separate detections of the fluorescent dyes. The optimal choice of excitation wavelengths and optical filters are discussed. The fluorphores that were used are Lucifer Yellow, Texas Red, fluorescein isothiocyanate and ... More
Chemistry of sulforhodamine--amine conjugates.
AuthorsCorrie JE, Davis CT, Eccleston JF
JournalBioconjug Chem
PubMed ID11312679
Commercially-available sulforhodamine sulfonyl chlorides contain two isomeric monosulfonyl chlorides. Conjugates of these isomers with amines have different properties because the sulfonamide formed from one isomer can undergo ring-closure to a colorless sultam. This chemistry has been examined for a model conjugate with methylamine and for a bioconjugate with 2'(3')-O-[N-(2-aminoethyl)carbamoyl]ATP. The ... More
Optical encoding with shaped DAM-1 molecular sieve particles.
AuthorsPantano P, Meek CC, Wang J, Coutinho DH, Balkus Jr KJ
JournalLab Chip
PubMed ID15100795
This work introduces a new high-throughput screening particle - a Dallas Amphorous Material No. 1 (DAM-1) molecular sieve particle. In contrast to porous silica microspheres, the 2-8-microm sized DAM-1 molecular sieve particles are available in a variety of shapes and morphologies including spheres, hexagons, rods, gyroids, and discoids. The advantage ... More
The synthesis of oligonucleotides containing an aliphatic amino group at the 5' terminus: synthesis of fluorescent DNA primers for use in DNA sequence analysis.
AuthorsSmith LM, Fung S, Hunkapiller MW, Hunkapiller TJ, Hood LE
JournalNucleic Acids Res
PubMed ID4000959
A rapid and versatile method has been developed for the synthesis of oligonucleotides which contain an aliphatic amino group at their 5' terminus. This amino group reacts specifically with a variety of electrophiles, thereby allowing other chemical species to be attached to the oligonucleotide. This chemistry has been utilized to ... More
Immunoassay techniques with fluorescent phycobiliprotein conjugates.
AuthorsKronick MN, Grossman PD
JournalClin Chem
PubMed ID6883673
We describe immunoassay techniques that take advantage of the novel properties of phycobiliprotein fluorescent dyes. These dyes, which can be isolated from a wide variety of algae, exhibit extremely high absorptivities, high quantum efficiencies, and excitation and emission bands across the visible spectrum. These stable, hydrophilic proteins can easily be ... More
Texas Red, a hydrophilic, red-emitting fluorophore for use with fluorescein in dual parameter flow microfluorometric and fluorescence microscopic studies.
AuthorsTitus JA, Haugland R, Sharrow SO, Segal DM
JournalJ Immunol Methods
PubMed ID6806389
The sulfonylchloride derivative of the red-emitting fluorophore, sulforhodamine 101, has been synthesized in order to provide a reagent for coupling to amino groups on proteins and other compounds, and it is now commercially available under the name 'Texas Red'. Texas Red conjugates of antibodies and other proteins have been prepared ... More
Chemical sensors based on controlled-release polymer systems.
AuthorsBarnard SM, Walt DR
JournalScience
PubMed ID2000492
A novel chemical sensor has been developed in which the polymer ethylene-vinyl acetate is used as a controlled-release system to deliver reagents to the sensing region of an optical fiber for a homogeneous competitive immunoassay based on fluorescence energy transfer. A competition reaction is used to enable continuous measurements of ... More
CCD microscopy and image analysis of cells and chromosomes stained by fluorescence in situ hybridization.
This paper reviews methods and applications of CCD microscopy for analysing cells and chromosomes subjected to fluorescence in situ hybridization (FISH). The current status of indirect and direct FISH staining methods with respect to probe labelling, detection sensitivity, multiplicity and DNA resolution is summarized. Microscope hardware, including special multi-band pass ... More
Simultaneous identification of eight leucocyte subsets of human peripheral blood using three-colour immunofluorescence flow cytometric analysis.
AuthorsCarayon P, Bord A, Raymond M
JournalJ Immunol Methods
PubMed ID2033277
The full analytical potential of flow cytometry has been exploited in order to identify a maximum of human peripheral blood leucocyte subsets in a single tube. For this purpose a mixture composed of six different monoclonal antibodies directly coupled to FITC or phycoerythrin or indirectly linked to the DuoChrome reagent ... More
Simultaneous on-line DNA sequencing on both strands with two fluorescent dyes.
AuthorsWiemann S, Stegemann J, Grothues D, Bosch A, Estivill X, Schwager C, Zimmermann J, Voss H, Ansorge W
JournalAnal Biochem
PubMed ID7710057
We describe an automated DNA-sequencing technique which allows both the simultaneous sequencing from the two strands of double-stranded templates and the subsequent detection of the sequencing products online and in parallel. The technique is based on hardware technology also used in the ALF DNA sequencer (Pharmacia, Uppsala). A helium-neon laser ... More
Which fluorophore is brightest? A comparison of the staining obtained using fluorescein, tetramethylrhodamine, lissamine rhodamine, Texas red, and cyanine 3.18.
AuthorsWessendorf MW, Brelje TC
JournalHistochemistry
PubMed ID1429023
There are several red-emitting fluorophores available for immunofluorescence studies, including tetramethylrhodamine, lissamine rhodamine, Texas Red, and cyanine 3.18; however, it is unclear which of these is best. The present study compared the brightness of these fluorophores to that of fluorescein. Staining was attempted using a primary antibody raised against serotonin ... More
Fluorescence spectroscopic profiling of compound libraries.
AuthorsSimeonov A, Jadhav A, Thomas CJ, Wang Y, Huang R, Southall NT, Shinn P, Smith J, Austin CP, Auld DS, Inglese J,
JournalJ Med Chem
PubMed ID18363325
Chromo/fluorophoric properties often accompany the heterocyclic scaffolds and impurities that comprise libraries used for high-throughput screening (HTS). These properties affect assay outputs obtained with optical detection, thus complicating analysis and leading to false positives and negatives. Here, we report the fluorescence profile of more than 70,000 samples across spectral regions ... More
Domain structure of the Staphylococcus aureus collagen adhesin.
Sequence analysis of surface proteins from Gram-positive bacteria indicates a composite organization consisting of unique and repeated segments. Thus, these proteins may contain discrete domains that could fold independently. In this paper, we have used a panel of biophysical methods, including gel permeation chromatography, analytical ultracentrifugation, circular dichroism, and fluorescence ... More
Energy transfer between two peptides bound to one MHC class II molecule.
AuthorsTampé R, Clark BR, McConnell HM
JournalScience
PubMed ID1656526
The 17-amino acid peptide from chicken ovalbumin, Ova(323-339), was labeled at the amino terminus with fluorescein [FOva(323-339)] and near the carboxyl terminus with Texas Red [AcOva(323-338)KTR]. Fluorescence spectroscopy was carried out on resolved electrophoretic bands on nonreducing polyacrylamide gels derived from incubation mixtures containing major histocompatibility complex (MHC) class II ... More
Probing the interaction between two single molecules: fluorescence resonance energy transfer between a single donor and a single acceptor.
We extend the sensitivity of fluorescence resonance energy transfer (FRET) to the single molecule level by measuring energy transfer between a single donor fluorophore and a single acceptor fluorophore. Near-field scanning optical microscopy (NSOM) is used to obtain simultaneous dual color images and emission spectra from donor and acceptor fluorophores ... More
Fast axonal transport of foreign synaptic vesicles in squid axoplasm.
AuthorsSchroer TA, Brady ST, Kelly RB
JournalJ Cell Biol
PubMed ID3848436
Translocation of intracellular organelles requires interaction with the cellular cytoskeleton, but the membrane and cytoskeletal proteins involved in movement are unknown. Here we show that highly purified synaptic vesicles from electric fish added to extruded squid axoplasm can show ATP-dependent movement. The movement is indistinguishable from that of endogenous vesicles ... More
Multiparameter analysis of human lymphocyte subpopulations using flow cytometry.
AuthorsLewis DE, Barron KS, Miller GP, Rich RR
JournalSurv Synth Pathol Res
PubMed ID2938244
This review has emphasized several recent advances in our understanding of human lymphocyte biology made possible by mAbs that recognize specific functional subpopulations of lymphocytes, coupled with multiparameter flow-cytometric analysis. In the last 3 years, both the T4+ and T8+ subsets of T cells have been further divided according to ... More
Three-color fluorescence measurements on single cells excited at three laser wavelengths.
AuthorsSteinkamp JA, Stewart CC, Crissman HA
JournalCytometry
PubMed ID7056131
A three-laser flow cytometer for quantitative analysis and sorting of cells and microscopic particles has been developed and evaluated using argon- and krypton-ion lasers as excitation sources. Cells stained with three fluorescent dyes having different excitation spectra enter a flow chamber where they first pass through an electronic volume sensor ... More
Inhibition of nucleolar reformation after microinjection of antibodies to RNA polymerase I into mitotic cells.
AuthorsBenavente R, Rose KM, Reimer G, Hügle-Dörr B, Scheer U
JournalJ Cell Biol
PubMed ID3312231
The formation of daughter nuclei and the reformation of nucleolar structures was studied after microinjection of antibodies to RNA polymerase I into dividing cultured cells (PtK2). The fate of several nucleolar proteins representing the three main structural subcomponents of the nucleolus was examined by immunofluorescence and electron microscopy. The results ... More
Synchronous luminescence: a new detection technique for multiple fluorescent probes used for DNA sequencing.
AuthorsStevenson CL, Johnson RW, Vo-Dinh T
JournalBiotechniques
PubMed ID8074876
A method fo nonradioactive DNA sequencing, which has been marketed commercially, uses four different fluorescent tags to label DNA fragments and fixed-wavelength excitation/fluorescence detection of the labels. This study presents an alternative method of detection based on synchronous luminescence (SL), in which both excitation and emission wavelengths are scanned simultaneously. ... More
Rapid screening and selection of monoclonal antibodies by bivariate flow cytometric analyses.
AuthorsPallavicini MG, Haendel S, Rosette C, Barlett R
JournalJ Immunol Methods
PubMed ID2464042
We describe a bivariate flow cytometric assay to rapidly identify hybridomas producing new monoclonal antibodies recognizing subpopulations that are unreactive with existing immunological reagents. In this screen, whole cells in microtiter wells are labeled first with a red-linked test antibody, and then with a green-linked cocktail of existing monoclonal antibody ... More
Leakage responses to L-NAME differ with the fluorescent dye used to label albumin.
AuthorsRumbaut RE, Harris NR, Sial AJ, Huxley VH, Granger DN
JournalAm J Physiol
PubMed ID9887048
Nitric oxide synthase (NOS) inhibitors have been reported to increase as well as to decrease microvascular transport of macromolecules in a variety of models. This study was performed to determine whether the influence of NOS inhibition on albumin leakage was dependent on the fluorescent dyes used to label albumin. Albumin ... More
A fluorescent study of ligands for guanidinobenzoatase, a protease associated with tumour cells.
AuthorsSteven F, Griffin MM, Williams LA, Feichter G
JournalAnticancer Res
PubMed ID3218954
We have employed ethanol-fixed wax embedded sections of human breast tumours and smears of rat leukaemia cells to provide test systems with recognisable tumour cells amongst normal cells. We have used 9-aminoacridine to locate cells possesing guanidinobenzoatase, an enzyme which degrades fibronectin and which binds 9-aminoacridine to its active centre. ... More
Differential partitioning of pulmonary surfactant protein SP-A into regions of monolayers of dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylcholine/dipalmitoylphosphatidylglycerol.
AuthorsRuano ML, Nag K, Worthman LA, Casals C, Pérez-Gil J, Keough KM
JournalBiophys J
PubMed ID9512012
The interaction of the pulmonary surfactant protein SP-A fluorescently labeled with Texas Red (TR-SP-A) with monolayers of dipalmitoylphosphatidylcholine (DPPC) and DPPC/dipalmitoylphosphatidylglycerol 7:3 w/w has been investigated. The monolayers were spread on aqueous subphases containing TR-SP-A. TR-SP-A interacted with the monolayers of DPPC to accumulate at the boundary regions between liquid ... More
Differential display with carboxy-X-rhodamine-labeled primers and the selection of differentially amplified cDNA fragments without cloning.
AuthorsYoshikawa Y, Mukai H, Asada K, Hino F, Kato I
JournalAnal Biochem
PubMed ID9466801
Differential display (DD) has been used extensively to detect differentially expressed genes. However, the low reproducibility of displayed bands makes verification by Northern blot difficult and the technique is labor-intensive. This report describes a fluorescent DD with a ROX (carboxy-X-rhodamine)-labeled anchor primer and a revised RT-PCR, utilizing AMV reverse transcriptase, ... More
Regulation of membrane type-1 matrix metalloproteinase activation by proprotein convertases.
AuthorsYana I, Weiss SJ
JournalMol Biol Cell
PubMed ID10888676
Membrane type-1 matrix metalloproteinase (MT1-MMP) is the prototypical member of a subgroup of membrane-anchored proteinases that belong to the matrix metalloproteinase family. Although synthesized as a zymogen, MT1-MMP plays an essential role in extracellular matrix remodeling after an undefined process that unmasks its catalytic domain. We now report the existence ... More
A modular detector for flow cytometric multicolor fluorescence measurements.
AuthorsSteinkamp JA, Habbersett RC, Stewart CC
JournalCytometry
PubMed ID3622158
A modular detector for measuring multicolor fluorescence from cells illuminated by single or multiple lasers has been developed for flow cytometers. Motion picture projector, camera, and CCTV/video lenses were evaluated for use in the detector by comparing their physical characteristics, image quality, and light collection efficiencies. A 25-mm focal length ... More
Differential intracellular staining of identified neurones in Locusta with texas red and lucifer yellow.
AuthorsSchneider H
JournalJ Neurosci Methods
PubMed ID2586149
The bright red fluorescent dye, Texas red, is introduced for ionophoretic microinjection in conjunction with the well-known dye Lucifer yellow. Different identified neurones can thus be visualised in two strongly contrasting colours in the same preparation (differential intracellular staining) following their physiological characterisation. Satisfactory results were obtained with electrodes filled ... More
Coexpression of T4 and T8 on peripheral blood T cells demonstrated by two-color fluorescence flow cytometry.
AuthorsBlue ML, Daley JF, Levine H, Schlossman SF
JournalJ Immunol
PubMed ID2982943
Using two-color fluorescence flow cytometry, we were able to detect the presence of small numbers of T4+T8+ cells (about 3%) in freshly isolated peripheral T cell populations derived from normal healthy donors. Coexpression of T4 and T8 was predominantly found on large blastlike cells and appeared to be related to ... More
Reconstitution of ATP-dependent movement of endocytic vesicles along microtubules in vitro: an oscillatory bidirectional process.
AuthorsMurray JW, Bananis E, Wolkoff AW
JournalMol Biol Cell
PubMed ID10679004
We have previously used the asialoglycoprotein receptor system to elucidate the pathway of hepatocytic processing of ligands such as asialoorosomucoid (ASOR). These studies suggested that endocytic vesicles bind to and travel along microtubules under the control of molecular motors such as cytoplasmic dynein. We now report reconstitution of this process ... More
A quantitative analysis of the retrograde axonal transport of 4 different fluorescent dyes in peripheral sensory and motor neurons and lack of anterograde transport in the corticospinal system.
AuthorsOgilvy CS, Borges LF
JournalBrain Res
PubMed ID2463856
Many fluorescent dye compounds are transported by axons in retrograde and anterograde directions. In the present study the uptake and retrograde axonal transport of 4 chemically related fluorescent dyes was evaluated in the peripheral nervous system of adult mice. Anterograde transport was studied in the corticospinal tract of adult rats. ... More
8 color, 10-parameter flow cytometry to elucidate complex leukocyte heterogeneity.
AuthorsRoederer M, De Rosa S, Gerstein R, Anderson M, Bigos M, Stovel R, Nozaki T, Parks D, Herzenberg L, Herzenberg L
JournalCytometry
PubMed ID9415416
We developed the chemistry, instrumentation, and software technologies needed to measure, simultaneously and independently, eight different fluorescent molecules on individual cells. Conjugation of these fluorochromes to monoclonal antibodies is straightforward; all immunofluorescence staining is accomplished with direct stains only. We built a hybrid flow cytometer with eight fluorescence detectors and ... More
Lateral electromigration and diffusion of Fc epsilon receptors on rat basophilic leukemia cells: effects of IgE binding.
AuthorsMcCloskey MA, Liu ZY, Poo MM
JournalJ Cell Biol
PubMed ID6236227
We have used in situ electromigration and post-field relaxation (Poo, M.-m., 1981, Annu. Rev. Biophys. Bioeng., 10:245-276) to assess the effect of immunoglobulin E (IgE) binding on the lateral mobility of IgE-Fc receptors in the plasmalemma of rat basophilic leukemia (RBL) cells. Bound IgE sharply increased the receptor's electrokinetic mobility, ... More
Nucleocytoplasmic shuttling: a novel in vivo property of antisense phosphorothioate oligodeoxynucleotides.
Phosphorothioate oligodeoxynucleotides (P=S ODNs) are frequently used as antisense agents to specifically interfere with the expression of cellular target genes. However, the cell biological properties of P=S ODNs are poorly understood. Here we show that P=S ODNs were able to continuously shuttle between the nucleus and the cytoplasm and that ... More
Functional methionines in the collagen/gelatin binding domain of plasma fibronectin: effects of chemical modification by chloramine T.
AuthorsMiles AM, Smith RL
JournalBiochemistry
PubMed ID8347617
Chemical modification of plasma fibronectin (pFn) or its 40-kDa collagen/gelatin binding (CGB) domain by low concentrations of chloramine T (CT), a methionine-specific oxidant, caused decreased binding affinity between pFn or the isolated CGB domain and Sepharose-immobilized denatured collagen or a Texas Red-labeled CNBr fragment CB7 from the alpha 1 chain ... More
Novel morphological approaches for the study of oocyte maturation.
AuthorsAlbertini DF
JournalBiol Reprod
PubMed ID6696960
Recent advances in light microscopy are discussed with respect to their application for the study of cell surface, cytoskeletal and organellar changes that occur during meiotic maturation in mammalian oocytes. Three techniques are considered: 1) multiple fluorochrome labeling using immunocytochemical or pharmacological probes to analyze the spatial and temporal disposition ... More
Fluorescence data analysis on gel-based biochips.
AuthorsBarsky V, Perov A, Tokalov S, Chudinov A, Kreindlin E, Sharonov A, Kotova E, Mirzabekov A
JournalJ Biomol Screen
PubMed ID12097187
A series of biochip readers developed for gel-based biochips includes three imaging models and a novel nonimaging biochip scanner. The imaging readers, ranging from a research-grade versatile reader to a simple portable one, use wide-field objectives and 12-bit digital large-coupled device cameras for parallel addressing of multiple array elements. This ... More
Griffonia simplicifolia lectins bind specifically to endothelial cells and some epithelial cells in mouse tissues.
AuthorsLaitinen L
JournalHistochem J
PubMed ID3597137
The binding of Griffonia simplicifolia agglutinin-I (GSA-I) and the isolectins GSA-I-AB3 and GSA-I-B4, having affinity for some alpha-D-galactosyl and N-acetyl galactosaminyl residues was studied in different mouse tissues. In brain, cardiac muscle and skeletal muscle, the GSA-I-lectin conjugates showed prominent binding only to blood vessel endothelia. Similarly, in the liver ... More
2,2'-thiodiethanol: a new water soluble mounting medium for high resolution optical microscopy.
AuthorsStaudt T, Lang MC, Medda R, Engelhardt J, Hell SW
JournalMicrosc Res Tech
PubMed ID17131355
The use of high numerical aperture immersion lenses in optical microscopy is compromised by spherical aberrations induced by the refractive index mismatch between the immersion system and the embedding medium of the sample. Especially when imaging >10 micro m deep into the specimen, the refractive index mismatch results in a ... More
Microtubule-dependent movement of late endocytic vesicles in vitro: requirements for Dynein and Kinesin.
AuthorsBananis E, Nath S, Gordon K, Satir P, Stockert RJ, Murray JW, Wolkoff AW
JournalMol Biol Cell
PubMed ID15181154
Our previous studies demonstrated that fluorescent early endocytic vesicles prepared from rat liver after injection of Texas red asialoorosomucoid contain asialoglycoprotein and its receptor and move and undergo fission along microtubules using kinesin I and KIFC2, with Rab4 regulating KIFC2 activity (J. Cell Sci. 116, 2749, 2003). In the current ... More
Solution-phase detection of polynucleotides using interacting fluorescent labels and competitive hybridization.
AuthorsMorrison LE, Halder TC, Stols LM
JournalAnal Biochem
PubMed ID2624314
DNA was assayed in a homogeneous format using DNA probes containing hybridization-sensitive labels. The DNA probes were prepared from complementary DNA strands in which one strand was covalently labeled on the 5'-terminus with fluorescein and the complementary strand was covalently labeled on the 3'-terminus with a quencher of fluorescein emission, ... More
Antigen-induced changes in B cell subsets in lymph nodes: analysis by dual fluorescence flow cytofluorometry.
AuthorsKraal G, Hardy RR, Gallatin WM, Weissman IL, Butcher EC
JournalEur J Immunol
PubMed ID2873041
Changes in the representation and surface phenotype of defined B cell subsets in murine lymph nodes stimulated with keyhole limpet hemocyanin or sheep red blood cells have been analyzed by two-color immunofluorescence fluorocytometric analysis. Shortly after immunization with either antigen there is a dramatic increase in both the frequency and ... More
Concomitant delineation of surface Ig, B-cell differentiation antigens, and HLADR on lymphoid proliferations using three-color immunocytometry.
AuthorsSegal GH, Edinger MG, Owen M, McNealis M, Lopez P, Perkins A, Linden MD, Fishleder AJ, Stoler MH, Tubbs RR
JournalCytometry
PubMed ID1712263
Accurate and consistent enumeration of B-cell subpopulations in lymphoid tissue was achieved through multiparameter three-color immunofluorescence and flow cytometric analysis (FCM). Phycoerythrin (PE)-anti-CD19 (Leu12) and biotinylated anti-HLADr/streptavidin-Duochrome (PE/Texas Red), used in conjunction with polyclonal fluorescein isothiocyanate (FITC) conjugated anti-surface immunoglobulin (SIg) antibodies, effectively separated non-specific binding and background fluorescence from ... More
Time-resolved, in vivo studies of mitotic spindle formation and nuclear lamina breakdown in Drosophila early embryos.
AuthorsPaddy MR, Saumweber H, Agard DA, Sedat JW
JournalJ Cell Sci
PubMed ID8907705
Time-resolved, two-component, three-dimensional fluorescence light microscopy imaging in living Drosophila early embryos is used to demonstrate that a large fraction of the nuclear envelope lamins remain localized to a rim in the nuclear periphery until well into metaphase. The process of lamin delocalization and dispersal, typical of 'open' forms of ... More
Fluorescence resonance energy transfer study of the associative state of membrane-bound complexes of complement proteins C5b-8.
AuthorsCheng KH, Wiedmer T, Sims PJ
JournalJ Immunol
PubMed ID3923109
Human complement protein C8 was labeled with the fluorescent chromophores fluorescein-5-isothiocyanate (FITC), 3-(4-isothiocyanatophenyl)-7-diethylamine-4-methyl coumarin (IPM), eosin-5-isothiocyanate (EOS), or Texas Red (sulforhodamine-101-sulfonyl chloride; TR) with only minor reduction in the specific hemolytic activity of the protein. The distribution of C5b-8 complexes bound to sheep erythrocyte membranes was investigated by monitoring fluorescence ... More
Evaluation of an internal positive control for Cryptosporidium and Giardia testing in water samples.
AuthorsWarnecke M, Weir C, Vesey G
JournalLett Appl Microbiol
PubMed ID12904227
AIMS: An internal positive control for Cryptosporidium and Giardia monitoring was evaluated for use in routine water monitoring quality control. The control, known as ColorSeed C&G (BTF Pty Ltd, Sydney, Australia), is a suspension containing exactly 100 Cryptosporidium oocysts and 100 Giardia cysts that have been modified by attachment of ... More
Human lymphocyte subpopulations identified by using three-color immunofluorescence and flow cytometry analysis: correlation of Leu-2, Leu-3, Leu-7, Leu-8, and Leu-11 cell surface antigen expression.
AuthorsLanier LL, Loken MR
JournalJ Immunol
PubMed ID6361119
Three-color immunofluorescence has been used to determine the co-expression of cell surface antigens on human peripheral blood lymphocytes. Monoclonal antibodies or avidin were coupled to either FITC (green), phycoerythrin (orange), or Texas Red (red) fluorochromes. These three fluorochromes could be independently measured by using a dual laser FACS IV system ... More
Voltage sensing by fluorescence resonance energy transfer in single cells.
AuthorsGonzález JE, Tsien RY
JournalBiophys J
PubMed ID8534797
A new mechanism has been developed for achieving fast ratiometric voltage-sensitive fluorescence changes in single cells using fluorescence resonance energy transfer. The mechanism is based on hydrophobic fluorescent anions that rapidly redistribute from one face of the plasma membrane to the other according to the Nernst equation. A voltage-sensitive fluorescent ... More
Characterization of hybridization between synthetic oligodeoxynucleotides and RNA in living cells.
AuthorsPolitz JC, Taneja KL, Singer RH
JournalNucleic Acids Res
PubMed ID8559650
Cells internalized synthetic oligonucleotides (oligos) in culture. The hybridization of these molecules to target RNA in the living cell was subsequently detected and characterized after fixation of the cells, with or without previous detergent extraction. Hybridized oligo was distinguished from free oligo in the cell using an in situ reverse ... More
Luminescence quenching by nitroxide spin labels in aqueous solution: studies on the mechanism of quenching.
AuthorsMatko J, Ohki K, Edidin M
JournalBiochemistry
PubMed ID1731926
The mechanism of luminescence quenching by spin labels was investigated in aqueous solution by steady-state and time-resolved luminescence techniques. Water-soluble nitroxide radicals strongly quenched the luminescence emitted by Tb3+ chelates and by fluorescein, either free or conjugated to proteins. The following features of the quenching reaction were established: (I) the ... More
Analysis of Fc (IgG) receptors on human peripheral blood leukocytes by dual fluorescence flow microfluorometry. II. Quantitation of receptors on cells that express the OKM1, OKT3, OKT4, and OKT8 antigens.
AuthorsTitus JA, Sharrow SO, Segal DM
JournalJ Immunol
PubMed ID6218201
Human peripheral blood mononuclear cells were analyzed for the expression of Fc(IgG) receptors (FcR) by a quantitative dual fluorescence flow microfluorometric (FMF) technique. Mononuclear cells from all donors tested were distributed into three distinct subsets on the basis of FcR expression: cells expressing high, intermediate, or background levels of FcR. ... More
The Golgi apparatus remains associated with microtubule organizing centers during myogenesis.
AuthorsTassin AM, Paintrand M, Berger EG, Bornens M
JournalJ Cell Biol
PubMed ID3894380
In vitro myogenesis involves a dramatic reorganization of the microtubular network, characterized principally by the relocalization of microtubule nucleating sites at the surface of the nuclei in myotubes, in marked contrast with the classical pericentriolar localization observed in myoblasts (Tassin, A. M., B. Maro, and M. Bornens, 1985, J. Cell ... More
Combinations of fluorescently labeled pulmonary surfactant proteins SP-B and SP-C in phospholipid films.
AuthorsNag K, Taneva SG, Perez-Gil J, Cruz A, Keough KM
JournalBiophys J
PubMed ID9168039
Hydrophobic pulmonary surfactant (PS) proteins B (SP-B) and C (SP-C) modulate the surface properties of PS lipids. Epifluorescence microscopy was performed on solvent-spread monolayers of fluorescently labeled porcine SP-B (R-SP-B, labeled with Texas Red) and SP-C (F-SP-C, labeled with fluorescein) in dipalmitoylphosphatidylcholine (DPPC) (at protein concentrations of 10 and 20 ... More
Single laser flow cytometric detection of lymphocytes binding three antibodies labelled with fluorescein, phycoerythrin and a novel tandem fluorochrome conjugate.
AuthorsFestin R, Björkland A, Tötterman TH
JournalJ Immunol Methods
PubMed ID2137494
In order to implement three-color surface immunofluorescence on a single laser flow cytometer, we combined a new fluorescent tandem conjugate (TC, R-phycoerythrin plus Texas red, available as DuoCHROME) with fluorescein (FITC)- and phycoerythrin (PE)-labelled monoclonal antibodies for the simultaneous detection of three antigens on individual lymphoid cells. Considerable amounts of ... More
Resonance energy transfer microscopy: observations of membrane-bound fluorescent probes in model membranes and in living cells.
AuthorsUster PS, Pagano RE
JournalJ Cell Biol
PubMed ID3771633
A conventional fluorescence microscope was modified to observe the sites of resonance energy transfer (RET) between fluorescent probes in model membranes and in living cells. These modifications, and the parameters necessary to observe RET between membrane-bound fluorochromes, are detailed for a system that uses N-4-nitrobenzo-2-oxa-1,3-diazole (NBD) or fluorescein as the ... More
Enhanced rotational dynamics of the phosphorylation domain of the Ca-ATPase upon calcium activation.
AuthorsHuang S, Squier TC
JournalBiochemistry
PubMed ID9922175
We have used labeling conditions that permit the specific and covalent attachment of erythrosin isothiocyanate (Er-ITC) to Lys464 within the phosphorylation domain of the Ca-ATPase in skeletal sarcoplasmic reticulum membranes. These labeling conditions do not interfere with high-affinity ATP binding, phosphoenzyme formation, or phosphoenzyme hydrolysis [Huang, S., Negash, S., and ... More
Fluorescence resonance energy transfer within a heterochromatic cAMP-dependent protein kinase holoenzyme under equilibrium conditions: new insights into the conformational changes that result in cAMP-dependent activation.
AuthorsJohnson DA, Leathers VL, Martinez AM, Walsh DA, Fletcher WH
JournalBiochemistry
PubMed ID8390856
Previous studies of the ligand regulation of the cAMP-dependent protein kinase have demonstrated the cAMP-mediated dissociation of the holoenzyme by using nonequilibrium techniques; i.e., gel filtration, ion-exchange chromatography, and differential centrifugation. While physically mild, these could have caused weakly associated species to dissociate, thereby providing a potentially flawed interpretation of ... More