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R P Haugland

Publications and source records attributed to R P Haugland.

At least 19 recordsLinked to original sources
PubMed2002

The ELF -97 phosphatase substrate provides a sensitive, photostable method for labelling cytological targets.

We compared fluorescent signals obtained with fluorescein conjugates and the ELF-97 (enzyme-labelled fluorescence) phosphatase substrate [2-(5'-chloro-2-phosphoryloxyphenyl)-6-chloro-4(3H)-quinazolinone] in labelling cytological structures requiring high spatial resolution. Enzymatic cleavage of the ELF-97 phosphatase substrate yields an extremely fine precipitate that remains well localized to the site of enzymatic activity. This precipitate fluoresces bright yellow-green, with maximal excitation at approximately 360 nm and maximal emission at approximately approximately 530 nm. The ELF substrate was used with streptavidin-alkaline phosphatase, to fluorescently label site-specific probes bound to their targets, including cell-surface sites, cytoplasmic organelles, nuclear antigens and cytoskeletal networks. All targets were labelled successfully with both the ELF substrate and fluoresceinated probes or protein conjugates. However, the ELF method was frequently more sensitive, with lower background fluorescence, allowing detection of more lysosomes, actin filaments, microtubules and nuclear targets than were visible with corresponding fluoresceinated probes. The ELF substrate was also used with antifluorescein-alkaline phosphatase to amplify fluorescein signals. We found that the ELF signal was in all cases brighter and more photostable than fluorescein signals, permitting shorter film exposures and allowing more time for examining samples. Surprisingly, relative brightness and photostability depended on the target, rather than being a general phenomenon related to the choice of dye alone.

3T3 Cells↗
PubMed2001

p75 and TrkA receptors are both required for uptake of NGF in adult sympathetic neurons: use of a novel fluorescent NGF conjugate.

We have developed and tested the biological activity and specificity of a novel fluorescent dextran-Texas Red-nerve growth factor (DTR-NGF) conjugate. DTR-NGF was found to promote survival and neurite outgrowth in cultured dissociated sympathetic neurons similarly to native NGF. The conjugate was taken up and transported retrogradely by terminal sympathetic nerves innervating the iris to neurons in the ipsilateral superior cervical ganglion (SCG) of young adult rats. Uptake and transport was assessed by counting numbers of labelled neurons and by measuring intensity of neuronal labelling using confocal microscopy and image analysis. DTR-NGF labelling in SCG neurons was shown to be dose-dependent with an EC(50) of 75 ng. Similar concentrations of unconjugated DTR resulted in no neuronal labelling. DTR-NGF uptake was competed off using a 50-fold excess of native NGF, resulting in a 73% reduction in numbers of labelled neurons. Pretreatment of nerve terminals with function-blocking antibodies against the low (p75) and high (TrkA) affinity NGF receptors resulted in a large (85-93%) reduction in numbers of DTR-NGF labelled neurons. Anti-p75 and anti-TrkA antibodies had comparable effects which were concentration-dependent. These findings indicate that both receptors are required for uptake of NGF in adult rat sympathetic neurons. In particular, the results provide strong evidence that the p75 receptor plays a more active role in transducing the NGF signal than has been proposed.

Animals↗
PubMed2001

Sensitive determination of cell number using the CyQUANT cell proliferation assay.

We describe here the development and characterization of the CyQUANT cell proliferation assay, a highly sensitive, fluorescence-based microplate assay for determining numbers of cultured cells. The assay employs CyQUANT GR dye, which produces a large fluorescence enhancement upon binding to cellular nucleic acids that can be measured using standard fluorescein excitation and emission wavelengths. The fluorescence emission of the dye-nucleic acid complexes correlated linearly with cell number over a large range using a wide variety of cell types. Under the recommended assay conditions, standard curves were linear (r(2)>0.995), detecting as few as 10-50 cells and as many as 25,000-50,000 cells with a single dye concentration, depending on cell type. Increasing the dye concentration extended the linear range of the assay to 100,000-250,000 cells. Results of cell proliferation and growth inhibition studies with the assay were similar to those obtained in published studies using other standard assays. CyQUANT assay measurements of serum-stimulated cell proliferation correlated well with measurements made using [3H]-thymidine. Also, the assay was used to analyze cellular DNA or RNA content, with the addition of a nuclease digestion step to the protocol. The assay procedure is simple and convenient, with no wash steps, and is readily amenable to automation.

3T3 Cells↗
PubMed2001

Green/red dual fluorescence detection of total protein and alkaline phosphate-conjugated probes on blotting membranes.

A two-color fluorescence detection method is described based upon covalently coupling the succinimidyl ester of BODIPY FL-X to proteins immobilized on poly(vinylidene difluoride) (PVDF) membranes, followed by detection of target proteins using the fluorogenic substrate 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl(DDAO)-phosphate in combination with alkaline-phosphatase-conjugated reporter molecules. This results in all proteins in the profile being visualized as green signal while those detected specifically with the alkaline-phosphatase conjugate appear as red signal. The dichromatic detection system is broadly compatible with a wide range of analytical imaging devices including UV epi- or transilluminators combined with photographic or charge-coupled device (CCD) cameras, xenon-arc sources equipped with appropriate excitation/emission filters, and dual laser gel scanners outfitted with a 473 nm second-harmonic generation or 488 nm argon-ion laser as well as a 633 nm helium-neon or 635 nm diode laser. The dichromatic detection method permits detection of low nanogram amounts of protein and allows for unambiguous identification of target proteins relative to the entire protein profile on a single electroblot, obviating the need to run replicate gels that would otherwise require visualization of total proteins by silver staining and subsequent alignment with chemiluminescent or colorimetric signals generated on electroblots.

Acridines↗
PubMed2001

Rapid and simple single nanogram detection of glycoproteins in polyacrylamide gels and on electroblots.

The fluorescent hydrazide, Pro-Q Emerald 300 dye, may be conjugated to glycoproteins by a periodic acid Schiff's (PAS) mechanism. The glycols present in glycoproteins are initially oxidized to aldehydes using periodic acid. The dye then reacts with the aldehydes to generate a highly fluorescent conjugate. Reduction with sodium metabisulfite or sodium borohydride is not required to stabilize the conjugate. Though glycoprotein detection may be performed on transfer membranes, direct detection in gels avoids electroblotting and glycoproteins may be visualized within 2-4 h of electrophoresis. This is substantially more rapid than PAS labeling with digoxigenin hydrazide followed by detection with an antidigoxigenin antibody conjugate of alkaline phosphatase, or PAS labeling with biotin hydrazide followed by detection with horseradish peroxidase or alkaline phosphatase conjugates of streptavidin, which require more than eight hours to complete. Pro-Q Emerald 300 dye-labeled gels and blots may be poststained with SYPRO Ruby dye, allowing sequential two-color detection of glycosylated and nonglycosylated proteins. Both fluorophores are excited with mid-range UV illumination. Pro-Q Emerald 300 dye maximally emits at 530 nm (green) while SYPRO Ruby dye maximally emits at 610 nm (red). As little as 300 pg of alpha 1-acid glycoprotein (40% carbohydrate) and 1 ng of glucose oxidase (12% carbohydrate) or avidin (7% carbohydrate) are detectable in gels after staining with Pro-Q Emerald 300 dye. Besides glycoproteins, as little as 2-4 ng of lipopolysaccharide is detectable in gels using Pro-Q Emerald 300 dye while 250-1000 ng is required for detection with conventional silver staining. Detection of glycoproteins may be achieved in sodium dodecyl sulfate-polyacrylamide gels, two-dimensional gels and on polyvinylidene difluoride membranes.

Animals↗
PubMed2000

Probing the cathepsin D using a BODIPY FL-pepstatin A: applications in fluorescence polarization and microscopy.

Redistribution of cathepsin D, a major lysosomal aspartic endopeptidase, has been related to various pathological progressions during tumor formation and oxidation stress. We have synthesized a fluorescent probe for cathepsin D, where the pepstatin A was covalently conjugated with the BODIPY (Boron dipyrromethene difluoride) fluorophore. In vitro, BODIPY FL-pepstatin A inhibits cathepsin D activity with an IC50 of 10 nM. The nature of its binding to cathepsin D was further characterized using a fluorescence polarization measurement. Results showed that BODIPY FL-pepstatin A selectively binds to cathepsin D at pH 4.5. In fixed cells, BODIPY FL-pepstatin A stained lysosomes, where it co-localized with cathepsin D. This staining was depleted when cells were co-incubated with unlabeled pepstatin A in acidic buffer. In live cells, BODIPY FL-pepstatin A is internalized and transported to lysosomes. The staining in the lysosomes can be competed with unlabeled pepstatin A. These properties, along with the good photostability of the BODIPY FL fluorophore, make this probe a novel tool for the study of the secretion and trafficking of cathepsin D.

Animals↗
PubMed2000

Ultrasensitive fluorescence protein detection in isoelectric focusing gels using a ruthenium metal chelate stain.

SYPRO Ruby IEF Protein Gel Stain is an ultrasensitive, luminescent stain optimized for the analysis of protein in isoelectric focusing gels. Proteins are stained in a ruthenium-containing metal complex overnight and then rinsed in distilled water for 2 h. Stained proteins can be excited by ultraviolet light of about 302 nm (UV-B transilluminator) or with visible light of about 470 nm. Fluorescence emission of the dye is maximal at approximately 610 nm. The sensitivity of the SYPRO Ruby IEF protein gel stain is superior to colloidal Coomassie blue stain and the highest sensitivity silver staining procedures available. The SYPRO Ruby IEF protein gel stain is suitable for staining proteins in nondenaturing or denaturing carrier ampholyte isoelectric focusing and immobilized pH gradient gel electrophoresis. The stain is compatible with N,N'-methylenebisacrylamide or piperazine diacylamide cross-linked polyacrylamide gels as well as with agarose gels and high tensile strength Duracryl gels. The stain does not contain extraneous chemicals (formaldehyde, glutaraldehyde, Tween-20) that frequently interfere with peptide identification in mass spectrometry. Successful identification of stained proteins by peptide mass profiling is demonstrated.

Amino Acid Sequence↗
PubMed2000

Background-free, high sensitivity staining of proteins in one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gels using a luminescent ruthenium complex.

SYPRO Ruby dye is a permanent stain comprised of ruthenium as part of an organic complex that interacts noncovalently with proteins. SYPRO Ruby Protein Gel Stain provides a sensitive, gentle, fluorescence-based method for detecting proteins in one-dimensional and two-dimensional sodium dodecyl sulfate-polyacrylamide gels. Proteins are fixed, stained from 3h to overnight and then rinsed in deionized water or dilute methanol/acetic acid solution for 30 min. The stain can be visualized using a wide range of excitation sources commonly used in image analysis systems including a 302 nm UV-B transilluminator, 473 nm second harmonic generation (SHG) laser, 488 nm argon-ion laser, 532 nm yttrium-aluminum-garnet (YAG) laser, xenon arc lamp, blue fluorescent light bulb or blue light-emitting diode (LED). The sensitivity of SYPRO Ruby Protein Gel Stain is superior to colloidal Coomassie Brilliant Blue (CBB) stain or monobromobimane labeling and comparable with the highest sensitivity silver or zinc-imidazole staining procedures available. The linear dynamic range of SYPRO Ruby Protein Gel stain extends over three orders of magnitude, which is vastly superior to silver, zinc-imidazole, monobromobimane and CBB stain. The fluorescent stain does not contain superfluous chemicals (formaldehyde, glutaraldehyde, Tween-20) that frequently interfere with peptide identification in mass spectrometry. While peptide mass profiles are severely altered in protein samples prelabeled with monobromobimane, successful identification of proteins by peptide mass profiling using matrix-assisted laser desorption/ionization mass spectrometry was easily performed after protein detection with SYPRO Ruby Protein Gel stain.

Dextrans↗
PubMed1999

Vitronectin interaction with glycosaminoglycans. Kinetics, structural determinants, and role in binding to endothelial cells.

Vitronectin (VN) is a high affinity heparin-binding protein. The physiological role of this binding has hitherto received little attention, and its molecular determinants are subject to controversy. In this study, we characterized vitronectin interaction with heparin, heparin analogues, bacterial extracts, and cell surface glycosaminoglycans. As assessed by (i) fluorescence assays, (ii) precipitation with heparin-Sepharose beads, or (iii) Western blotting with antibodies against VN(347-361) (the heparin-binding site), we demonstrate an exposure of the VN heparin-binding site in multimeric but not monomeric vitronectin. Through its heparin-binding site, vitronectin also bound other glycosaminoglycans and Staphylococcus aureus extracts. The kinetics of heparin binding to vitronectin were complex. After a fast association phase (tau = 0.3 s), a slow conversion of an unstable to a stable heparin-vitronectin complex (tau = 180 s) occurred. Heparin binding kinetics and transition to a stable complex were mimicked by VN(347-361), demonstrating that this area is the fully functional heparin-binding site of vitronectin. Multimeric vitronectin bound to endothelial cells. This binding was blocked by soluble heparin and was not observed when endothelial cells were pretreated with glycosaminoglycan-removing enzymes. Glycosaminoglycan-dependent interaction of endothelial cells with multimeric vitronectin might be a relevant mechanism for removal of multimeric vitronectin from plasma. Conversion of an unstable to a stable glycosaminoglycan-vitronectin complex is likely to be relevant for association with endothelial cells under flow conditions.

Cells, Cultured↗
PubMed1999

A luminescent ruthenium complex for ultrasensitive detection of proteins immobilized on membrane supports.

SYPRO Ruby protein blot stain provides a sensitive, gentle, fluorescence-based method for detecting proteins on nitrocellulose or polyvinylidene difluoride (PVDF) membranes. SYPRO Ruby dye is a permanent stain composed of ruthenium as part of an organic complex that interacts noncovalently with proteins. Stained proteins can be excited by ultraviolet light of about 302 nm or with visible light of about 470 nm. Fluorescence emission of the dye is approximately 618 nm. The stain can be visualized using a wide range of excitation sources utilized in image analysis systems including a UV-B transilluminator, 488-nm argon-ion laser, 532-nm yttrium-aluminum-garnet (YAG) laser, blue fluorescent light bulb, or blue light-emitting diode (LED). The detection sensitivity of SYPRO Ruby protein blot stain (0.25-1 ng protein/mm(2)) is superior to that of amido black, Coomassie blue, and india ink staining and nearly matches colloidal gold staining. SYPRO Ruby protein blot stain visualizes proteins more rapidly than colloidal gold stain and the linear dynamic range is more extensive. Unlike colloidal gold stain, SYPRO Ruby protein blot stain is fully compatible with subsequent biochemical applications including colorimetric and chemiluminescent immunoblotting, Edman-based sequencing and mass spectrometry.

Collodion↗
PubMed1999

Fluorogenic substrates based on fluorinated umbelliferones for continuous assays of phosphatases and beta-galactosidases.

Fluorogenic substrates based on 4-methylumbelliferone (4-MU) have been widely used for the detection of phosphatase and glycosidase activities. One disadvantage of these substrates, however, is that maximum fluorescence of the reaction product requires an alkaline pH, since 4-MU has a pK(a) approximately 8. In an initial screening of five phosphatase substrates based on fluorinated derivatives of 4-MU, all with pK(a) values lower than that of 4-MU, we found that one substrate, 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP), was much improved for the detection of acid phosphatase activity. When measured at the preferred acid phosphatase reaction pH (5.0), DiFMUP yielded fluorescence signals that were more than 10-fold higher than those of 4-methylumbelliferyl phosphate (MUP). DiFMUP was also superior to MUP for the detection of protein phosphatase 1 activity at pH 7 and was just as sensitive as MUP for the detection of alkaline phosphatase activity at pH 10. A beta-galactosidase substrate was also prepared based on 6, 8-difluoro-4-methylumbelliferone. This substrate, 6, 8-difluoro-4-methylumbelliferyl beta-d-galactopyranoside (DiFMUG), was found to be considerably more sensitive than the commonly used substrate 4-methylumbelliferyl beta-d-galactopyranoside (MUG), for the detection of beta-galactosidase activity at pH 7. DiFMUP and DiFMUG should have great utility for the continuous assay of phosphatase and beta-galactosidase activity, respectively, at neutral and acid pH.

Acid Phosphatase↗
PubMed1999

5-(Pentafluorobenzoylamino)fluorescein: A selective substrate for the determination of glutathione concentration and glutathione S-transferase activity.

5-(Pentafluorobenzoylamino)fluorescein (PFB-F), a new thiol-reactive molecule was synthesized to improve the detection limits and specificity of the assays for glutathione S-transferase (GST) activity and glutathione (GSH). A rapid assay method to measure GSH concentration or GST activity and the simultaneous analysis of multiple samples is possible because the glutathione adduct, GS-TFB-F, is separated from PFB-F by thin-layer chromatography (TLC) and can be quantitated by a fluorescence scanner. The detection limits for GSH and for GST activity using TLC were found to be as low as 10 pmol/microl and 1 ng/microl using equine liver GST, respectively. Determination of GSH concentration or GST activity in bovine pulmonary artery endothelial (BPAE) cell lysates gave a linear response for samples corresponding to 500-2500 cells. PFB-F could also measure GST activities of GST fusion proteins and prove to be a suitable substrate for determining the activities of human GST isozymes and other sources of mammalian GST. The selectivity of PFB-F with GSH was proven by comparing trace amount of the adducts that formed with cysteine and beta-galactosidase to that formed with GSH. The HPLC profile of a reaction mixture where cell lysate was used in place of purified GST, also shows only two main peaks, corresponding to GS-TFB-F and unreacted PFB-F. The selectivity of PFB-F for GSH was further confirmed by exposing BPAE cells to dl-buthionine-[S,R]-sulfoximine (BSO). Our results of GS-TFB-F determination indicate that 12-, 24-, or 36-h incubations with BSO caused 2-, 6-, or 7.6-fold reductions in GSH levels, respectively.

Animals↗
PubMed1999

A novel acidotropic pH indicator and its potential application in labeling acidic organelles of live cells.

BACKGROUND: Ratio imaging has received intensive attention in the past few decades. The growing potential of ratio imaging is significantly limited, however, by the lack of appropriate fluorescent probes, for acidic organelles in particular. The classic fluorescent dyes (such as fluoresceins, rhodamines and coumarins) are not suitable for studying acidic organelles (such as lysosomes) because their fluorescence is significantly decreased under neutral or acidic conditions. This has motivated us to develop probes that can be used in ratio imaging that are strongly fluorescent even in acidic media. RESULTS: The compound 2-(4-pyridyl)-5-((4-(2-dimethylaminoethyl-aminocarbamoyl) methoxy)phenyl)oxazole (PDMPO) was prepared and characterized as a new acidotropic dual-excitation and dual-emission pH indicator. It emits intense yellow fluorescence at lower pH and gives intense blue fluorescence at higher pH. This unique pH-dependent fluorescence property was readily explored to selectively stain lysosomes and to determine the pH of the organelle in an emission-ratio-imaging mode. PDMPO is selectively localized to lysosomes and exhibits a pH-dependent dual excitation and emission. CONCLUSIONS: PDMPO selectively labels acidic organelles (such as lysosomes) of live cells and the two distinct emission peaks can be used to monitor the pH fluctuations of live cells in ratio measurements. Additionally, the very large Stokes shift and excellent photostability of PDMPO make the compound an ideal fluorescent acidotropic probe. The unique fluorescence properties of PDMPO might give researchers a new tool with which to study acidic organelles of live cells.

Acids↗
PubMed1999

Alexa dyes, a series of new fluorescent dyes that yield exceptionally bright, photostable conjugates.

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 Alexa names indicate the approximate excitation wavelength maximum in nm). All Alexa dyes and their conjugates are more fluorescent and more photostable than their commonly used spectral analogues listed above. In addition, Alexa dyes are insensitive to pH in the 4-10 range. We evaluated Alexa dyes compared with conventional dyes in applications using various conjugates, including those of goat anti-mouse IgG (GAM), streptavidin, wheat germ agglutinin (WGA), and concanavalin A (ConA). Conjugates of Alexa 546 are at least twofold more fluorescent than Cy3 conjugates. Proteins labeled with the Alexa 568 or Alexa 594 dyes are several-fold brighter than the same proteins labeled with lissamine rhodamine B or Texas Red dyes, respectively. Alexa dye derivatives of phalloidin stain F-actin with high specificity. Hydrazide forms of the Alexa dyes are very bright, formaldehyde-fixable polar tracers. Conjugates of the Alexa 430 (ex 430 nm/em 520 nm) and Alexa 532 (ex 530 nm/em 548 nm) fluorochromes are spectrally unique fluorescent probes, with relatively high quantum yields in their excitation and emission wavelength ranges.

Animals↗
PubMed1998

Pairs of violet-light-excited fluorochromes for flow cytometric analysis.

We describe pairs of fluorochromes for use with the 407-nm line of a violet-light-enhanced krypton ion laser. These fluorochromes and a previously described violet-light-excited reporter variant, GFP-Vex, fall into two emission classes: blue for Cascade Blue, and green/yellow for Cascade Yellow, Lucifer Yellow, and GFP-Vex. Cascade Yellow is a new fluorochrome that we have synthesized and is used for the first time in the present study. The two emission classes are sufficiently different that Cascade Blue can be paired with Cascade Yellow, Lucifer Yellow, or GFP-Vex in flow cytometric analysis. Furthermore, with proper detection filters, these fluorochromes can be combined with all of the currently used fluorochromes in a three-laser FACS system. With these data, the total number of fluorochromes that can be used as antibody labels for simultaneous detection in combined FACS analysis increases to nine. This study demonstrates the sensitivity and power of the combined use of these reagents in a single eight-color analysis by identifying murine T-lymphocyte subsets that could not otherwise be readily distinguished.

Animals↗
PubMed1998

Synthesis of novel fluorinated coumarins: excellent UV-light excitable fluorescent dyes.

Two new fluorinated fluorescent dyes, 6,8-difluoro-7-hydroxy-4-methylcoumarin (Marina Blue) and 3-carboxy-6,8-difluoro-7-hydroxycoumarin (Pacific Blue), exhibit excellent photophysical properties among a series of novel fluorinated 7-hydroxycoumarins. Most of these fluorinated coumarins have quantum yields (0.63 to 0.89) equal to or higher than that of the parent compound (0.63), which, in combination with their lower pKaS and higher photostability, make them superior fluorescent dyes for use as reporter molecules in biological systems.

Coumarins↗
PubMed1998

Novel flow cytometry compensation standards: internally stained fluorescent microspheres with matched emission spectra and long-term stability.

In flow cytometry, the emission spectral overlap of fluorescein and R-phycoerythrin is usually corrected by electronic color compensation using microspheres surface labeled with the same fluorochromes. However, the inherent chemical instability of these fluorochromes may cause inaccurate compensation. To overcome these problems, Compen-Flow beads, a new type of compensation standards were developed. The CompenFlow beads are a set of 6.0-microm-diameter polystyrene microspheres that are internally stained with selected BODIPY dye combinations. When excited by the 488-nm argon laser line, these beads show a nearly perfect emission spectral match to fluorescein-stained, R-phycoerythrin-stained and unstained lymphocytes, respectively. Moreover, since the dye molecules are oil soluble, they are contained inside the microsphere matrix instead of merely on the surface; thus, the molecules are shielded from environmental factors that could affect an exposed fluorochrome. Our results show a stable fluorescence spectral profile and constant intensity for at least 2 years stored either refrigerated or at room temperature.

Drug Stability↗