Rapid staining of proteins on polyacrylamide gels and nitrocellulose membranes using a mixture of fluorescent dyes

The present work describes a novel, fluorescence-based method for staining proteins on SDS-PAGE and membrane(s). In this method, proteins are stained using a mixed-dye (sulfo-rhodamine B and 1-anilino-8-naphthalene sulfonic acid (NH(4)(+))) solution. The mixed-dye staining protocol can detect proteins up to a concentration of 15 ng. This method is generally applicable to all proteins and is more sensitive than the conventional Coomassie blue method. The staining method is rapid and efficient. Staining-destaining of proteins using the mixed-dye protocol takes less than half an hour. Another interesting feature of the staining protocol described here is the applicability to the staining of proteins on nitrocellulose membranes.     

Ultrafast coelectrophoretic fluorescent staining of proteins with carbocyanines

Protein detection on SDS gels or on 2-D gels must combine several features, such as sensitivity, homogeneity from one protein to another, speed, low cost, and user-friendliness. For some applications, it is also interesting to have a nonfixing stain, so that proteins can be mobilized from the gel for further use (electroelution, blotting). We show here that coelectrophoretic staining by fluorophores of the oxacarbocyanine family, and especially diheptyloxacarbocyanine, offers several positive features. The sensitivity is intermediate between the one of colloidal CBB and the one of fluorescent ruthenium complexes. Detection is achieved within 1 h after the end of the electrophoretic process and does not use any fixing or toxic agent. The fluorescent SDS-carbocyanine-protein complexes can be detected either with a laser scanner with an excitation wavelength of 488 nm or with a UV table operating at 302 nm. Excellent sequence coverage in subsequent MS analysis of proteolytic peptides is also achieved with this detection method.     

Alternate gram staining technique using a fluorescent lectin.

Fluorescence-labeled wheat germ agglutinin binds specifically to N-acetylglucosamine in the outer peptidoglycan layer of gram-positive bacteria. The peptidoglycan layer of gram-negative bacteria is covered by a membrane and is not labeled by the lectin. By exploiting this phenomenon, an alternative Gram staining technique has been developed.     

Alternate gram staining technique using a fluorescent lectin.

Fluorescence-labeled wheat germ agglutinin binds specifically to N-acetylglucosamine in the outer peptidoglycan layer of gram-positive bacteria. The peptidoglycan layer of gram-negative bacteria is covered by a membrane and is not labeled by the lectin. By exploiting this phenomenon, an alternative Gram staining technique has been developed.     

Direct genomic fluorescent on-line sequencing and analysis using in vitro amplification of DNA.

In vitro amplification of genomic DNA and total RNA, as well as recombinant DNA, using one fluorescently labelled and one unlabelled primer during amplification, together with on-line analysis of the products on the EMBL fluorescent DNA sequencer, is described. Further is reported direct sequencing of fluorescently labelled amplified probes by solid-phase chemical degradation, without subcloning and purification steps involved. At present up to 350 bases in 4 hours are determined with this technique. The fluorescent dye and its bond to the oligonucleotide are stable during the amplification cycles, and do not interfere with the enzymatic polymerization. High sensitivity of the detection device, down to 10(-18) moles, corresponding to less than 10(6) molecules makes possible analyses of the non-radioactive amplified probes after only 10 amplification cycles, starting with about 5 x 10(4) copies of recombinant DNA.     

Visible fluorescent detection of proteins in polyacrylamide gels without staining

2,2,2-Trichloroethanol (TCE) incorporated into polyacrylamide gels before polymerization provides fluorescent visible detection of proteins in less than 5min of total processing time. The tryptophans in proteins undergo an ultraviolet light-induced reaction with trihalocompounds to produce fluorescence in the visible range so that the protein bands can be visualized on a 300-nm transilluminator. In a previous study trichloroacetic acid or chloroform was used to stain polyacrylamide gel electrophoresis (PAGE) gels for protein visualization. This study shows that placing TCE in the gel before electrophoresis can eliminate the staining step. The gel is removed from the electrophoresis apparatus and placed on a transilluminator and then the protein bands develop their fluorescence in less than 5min. In addition to being rapid this visualization method provides detection of 0.2microg of typical globular proteins, which for some proteins is slightly more sensitive than the standard Coomassie brilliant blue (CBB) method. Integral membrane proteins, which do not stain well with CBB, are visualized well with the TCE in-gel method. After TCE in-gel visualization the same gel can then be CBB stained, allowing for complementary detection of proteins. In addition, visualization with TCE in the gel is compatible with two-dimensional PAGE, native PAGE, Western blotting, and autoradiography.     

Direct measurement of antigen binding properties of CD1 proteins using fluorescent lipid probes

CD1 proteins are antigen-presenting molecules that bind foreign and self-lipids and stimulate specific T cell responses. In the current study, we investigated ligand binding by CD1 proteins by developing a fluorescent probe binding approach using soluble recombinant human CD1 proteins. To increase stability and yield, soluble group 1 CD1 (CD1b and CD1c) and group 2 CD1 (CD1d) proteins were produced as single chain secreted CD1 proteins in which beta2-microglobulin was fused to the N termini of the CD1 heavy chains by a flexible peptide linker sequence. Analysis of ligand binding properties of single chain secreted CD1 proteins by using fluorescent lipid probes indicated significant differences in ligand preference and in pH dependence of binding by group 1 versus group 2 CD1 proteins. Whereas group 1 CD1 isoforms (CD1b and CD1c) show stronger binding of nitrobenzoxadiazole (NBD)-labeled dialkyl-based ligands (phosphatidylcholine, sphingomyelin, and ceramide), group 2 CD1 (CD1d) proteins were stronger binders of small hydrophobic probes such as 1-anilinonaphthalene-8-sulfonic acid and 4,4'-dianilino-1,1'-naphthyl-5,5'-disulfonic acid. Competition studies indicated that binding of fluorescent lipid probes involved association of the probe with the hydrophobic ligand binding groove of CD1 proteins. Analysis of selected alanine substitution mutants of human CD1b known to inhibit antigen presentation showed that NBD-labeled lipid probe binding could be used to distinguish mutations that interfere with ligand binding from those that affect T cell receptor docking. Our findings provide further evidence for the functional specialization of different CD1 isoforms and demonstrate the value of the fluorescent lipid probe binding method for assisting structure-based studies of CD1 function.     

pH sensing in living cells using fluorescent microspheres

Intracellular pH in living cells is measured in real time at the single cell level using fluorescently covalently loaded microspheres as efficient carrier systems and stable sensors. The use of these sensors immobilized covalently onto polymeric particles allows analysis of intracellular pH flux over long period of time and eliminates the disadvantages such as dilution within the cell, elimination via leakage or compartmentalization.     

Multiplexed molecular interactions of nuclear receptors using fluorescent microspheres

BACKGROUND: We describe a novel microsphere-based system to identify and characterize multiplexed interactions of nuclear receptors with peptides that represent the LXXLL binding region of coactivator proteins. METHODS: In this system, individual microsphere populations with unique red and orange fluorescent profiles are coupled to specific coactivator peptides. The coactivator peptide-coupled microsphere populations are combined and incubated with a nuclear receptor that has been coupled to a green fluorochrome. Flow cytometric analysis of the microspheres simultaneously decodes each population and detects the binding of receptor to respective coactivator peptides by the acquisition of green fluorescence. RESULTS: We have used this system to determine the binding affinities of human estrogen receptor beta ligand binding domain (ERbeta LBD) and human peroxisome proliferator activated receptor gamma ligand binding domain (PPARgamma LBD) to a set of 34 coactivator peptides. Binding of ERbeta LBD to a coactivator peptide sequence containing the second LXXLL motif of steroid receptor coactivator-1 (SRC-1(2) (676-700) is shown to be specific and saturable. Analysis of receptor binding to a multiplexed set of coactivator peptides shows PPARgamma LBD binds with high affinity to cAMP response element binding protein (CBP) peptides and to the related P300 peptide while ERbeta LBD exibits little binding to these peptides. Using the microsphere-based assay we demonstrate that ERbeta LBD and PPARgamma LBD binding affinities for the coactivator peptides are increased in the presence of agonist (estradiol or GW1929, respectively) and that ERbeta LBD binding is decreased in the presence of antagonist (raloxifene or tamoxifen). CONCLUSIONS: This unique microsphere-based system is a sensitive and efficient method to simultaneously evaluate many receptor-coactivator interactions in a single assay volume. In addition, the system offers a powerful approach to study small molecule modulation of nuclear receptor binding.     

Detection of low-density cell-surface molecules using biotinylated fluorescent microspheres

Biotinylated fluorescent microspheres have been developed as a reagent for studying antigens and receptors expressed at the cell surface. Labeling of antigen or receptor was accomplished by crosslinking biotinylated microspheres through streptavidin to corresponding biotinylated antibodies or ligands. Detection of labeled cells by flow microfluorimetry provided an extremely sensitive means for the analysis and potential manipulation of heterogeneous cell populations. The data indicate that cells bearing fewer than 200 surface antigen-antibody complexes per cell are readily detectable by this approach. Crosslinked to a selected biotinylated peptide immunogen, biotinylated fluorescent microspheres also allowed the labeling and detection of hybridoma cells bearing antigen-specific surface immunoglobulin.     

Direct assessment and distribution of regional portal blood flow in the pig by means of fluorescent microspheres.

Measurement of regional organ blood flow by means of fluorescent microspheres (FM) is an accepted method. However, determination of regional portal blood flow (RPBF) cannot be performed by microspheres owing to the entrapment of the spheres in the upstream capillary bed of the splanchnic organs. We hypothesized that an adequate experimental setting would enable us to measure RPBF by means of FM and to analyze its distribution within the pig liver. A mixing chamber for the injection of FM was developed, and its capability to distribute FM homogeneously in the blood was evaluated in vitro. The chamber was implanted into the portal vein of six anesthetized pigs (23.5 +/- 2.9 kg body wt). Three consecutive, simultaneous injections of FM of two different colors into the chamber were performed. Reference portal blood samples were collected by means of a Harvard pump. At the end of the experiment, the liver was explanted and fixed in formalin before dissection. FM were isolated from the tissue samples by an automated process, and fluorescence intensity was determined. Comparison of 5,458 single RPBF values, determined by simultaneously injected FM, revealed good agreement (bias 2.5%, precision 12.7%) and high correlation (r = 0.97, r2 = 0,95, slope = 1.04, intercept = 0.05). Median RPBF was 1.07 +/- 0.78 ml x min(-1) x g(-1). Allocation of the blood flow values to the anatomic regions of the liver revealed a significantly higher RPBF (P = 0.01) in the liver tissue located close to the diaphragm compared with the rest of the organ and a significantly lower RPBF (P = 0.01) in the left liver lobe compared with the median and right lobes. The results show that the model presented makes it possible to measure RPBF by means of FM reliably and that RPBF is distributed heterogeneously in the porcine liver.     

Direct and indirect fluorescent antibody staining ofOphiobolus graminis Sacc. in culture and in the rhizosphere of cereal plants

Fluorescein isothiocyanate (FITC) conjugates were prepared to whole-cell and cell-wall fractions of four cultures ofOphiobolus graminis W1, W2, O1 and O₂. Homologous and heterologous direct and indirect FA staining of the four cultures ofO. graminis gave positive staining in all reactions. This indicated that the four cultures could not be differentiated by fluorescent antibody (FA) staining. Species specificity of the conjugates was shown by the staining ofO. graminis hyphae in the rhizosphere of wheat and oat roots. Plant tissues were not stained. Furthermore out of 52 rhizosphere isolates stained with whole-cell and cell-wall conjugates of the four cultures ofO. graminis, only 7 cross-reacted with the whole-cell conjugates whereas none cross-reacted with the cell-wall conjugates.These results indicate the potentiality of the FA staining technique as a serological tool in localizing, and identifyingO. graminis amongst mixed fungal populations in the rhizosphere of roots.     

Assessment of spermatozoal function using dual fluorescent staining and flow cytometric analyses

Spermatozoa from bulls, boars, dogs, horses, mice, and men were examined using a fluorogenic stain consisting of the membrane-permeant substrate carboxyfluorescin diacetate (CFDA) and the relatively membrane-impermeant nuclear stain propidium iodide (PI). Three distinct populations of spermatozoa were discernible in samples from each species upon microscopic examination. Individual spermatozoa, presumed to be viable because of their motility, retained products of the fluorescein chromophore throughout the cell. A second population of spermatozoa in which the nuclei stained red with PI retained the green fluorescein fluorophore mainly in the acrosome. A third population, presumed to be degenerate spermatozoa, possessed only red fluorescent nuclei. These populations were quantified using dual parameter flow cytometry in 14 samples of cryopreserved bovine spermatozoa for which fertility and seminal quality data were available. Flow cytometric analyses were highly correlated with other seminal quality measurements. Sequential flow cytometric analyses provided the ability to rapidly quantitate changes in specific fluorescently stained populations. The ability to make rapid quantitative measurements should allow development of new and presumably more reliable information on the functional aspects of spermatozoa.     

Detection of areas of wall differentiation in fungi using fluorescent staining

Fungal colonies were stained with a fluorescent brightner, Calcofluor White M2R New. When viewed using ultraviolet light certain hyphal regions showed intense fluorescence, whereas others showed a lower intensity fluorescence. The apical 10 μm of leadingBotrytis cinerea hyphae showed intense fluorescence. Developing septa and side branches also showed this intense localised fluorescence. Calcofluor staining of hyphae and their subsequent growth illustrated the phenomenon of tip extension.     

Combination of silver and fluorescent staining for metaphase chromosomes.

A convenient and reliable method for simulatneous visualization of silver staining (Ag-NOR) of the nucleolus organizers and fluorescent bandings in metaphase chromosomes is described. Studies employing this combined procedure on human chromosomes revealed that the Ag-NOR patterns may be characteristic for each chromosome of each individual.     

利用荧光染色和流式细胞技术辅助卵孢小奥德蘑原生质体制备与再生研究

本研究以卵孢小奥德蘑液体培养菌丝作为实验材料,利用单因子变量法探索研究了菌丝培养时间、酶浓度、酶解时间、酶解温度、稳渗剂类型对卵孢小奥德蘑原生质体制备的影响,并对原生质体再生培养基进行选择和优化。通过荧光染色,利用激光共聚焦显微镜和流式细胞仪对原生质体的制备过程、得率和活力进行研究。结果表明,将卵孢小奥德蘑菌丝在液体培养基中培养5d收集菌丝体,以甘露醇作为渗透压稳定剂,在溶壁酶浓度2%、30℃条件下酶解5h,获得的原生质体得率最高,达2.0×10 ⁷个/mL;通过流式细胞仪分析,约57.69%的原生质体细胞为活细胞;在RM培养基中再生效果最好,再生率为(0.103±0.025)%。研究结果可以为卵孢小奥德蘑育种与食用菌原生质体制备再生提供研究基础。