Interaction of fluorescently-labeled contractile proteins with the cytoskeleton in cell models

To determine if a living cell is necessary for the incorporation of actin, alpha-actinin, and tropomyosin into the cytoskeleton, we have exposed cell models to fluorescently labeled contractile proteins. In this in vitro system, lissamine rhodamine-labeled actin bound to attachment plaques, ruffles, cleavage furrows and stress fibers, and the binding could not be blocked by prior exposure to unlabeled actin. Fluorescently labeled alpha-actinin also bound to ruffles, attachment plaques, cleavage furrows, and stress fibers. The periodicity of fluorescent alpha-actinin along stress fibers was wider in gerbil fibroma cells than it was in PtK2 cells. The fluorescent alpha-actinin binding in cell models could not be blocked by the prior addition of unlabeled alpha-actinin suggesting that alpha-actinin was binding to itself. While there was only slight binding of fluorescent tropomyosin to the cytoskeleton of interphase cells, there was stronger binding in furrow regions of models of dividing cells. The binding of fluorescently labeled tropomyosin could be blocked by prior exposure of the cell models to unlabeled tropomyosin. If unlabeled actin was permitted to polymerize in the stress fibers in cell models, fluorescently labeled tropomyosin stained the fibers. In contrast to the labeled contractile proteins, fluorescently labeled ovalbumin and BSA did not stain any elements of the cytoskeleton. Our results are discussed in terms of the structure and assembly of stress fibers and cleavage furrows.     

Cell microarrays: an emerging technology for the characterization of antibodies

The possibility to miniaturize and parallelize biological assays has a great impact on the development of biomedical technologies. Here, we describe a simple, miniaturized, and parallelized method employing entire cells from different cell lines displaying a protein of interest on their surface, which were immobilized on a microarray slide. Antibodies were added to these cellular microarrays, and their specific binding to the cell surface proteins was monitored using appropriate fluorescently labeled detection molecules. This new method is applicable for rapidly screening cell surface-specific antibodies with respect to selectivity and cross-reactivity.     

Serum microarrays for large scale screening of protein levels

There is a great need for comprehensive proteomic analysis of large patient cohorts of plasma and serum samples to identify biomarkers of human diseases. Here we describe a new antibody-based proteomic approach involving a reverse array format where serum samples are spotted on a microarray. This enables all samples to be screened for their content of a certain serum protein in a single experiment using target-recognizing antibodies and fluorescently labeled secondary antibodies. The procedure is illustrated with the analysis of the IgA levels in 2009 spotted serum samples, and the data are compared with clinical routine measurements. The results suggest that it is possible to simultaneously screen thousands of complex clinical serum samples for their content of the relative amount of specific serum proteins of clinical relevance.     

Fluorescent labeling in semi-solid medium for selection of mammalian cells secreting high-levels of recombinant proteins

Background  

Despite the powerful impact in recent years of gene expression markers like the green fluorescent protein (GFP) to link the expression of recombinant protein for selection of high producers, there is a strong incentive to develop rapid and efficient methods for isolating mammalian cell clones secreting high levels of marker-free recombinant proteins. Recently, a method combining cell colony growth in methylcellulose-based medium with detection by a fluorescently labeled secondary antibody or antigen has shown promise for the selection of Chinese Hamster Ovary (CHO) cell lines secreting recombinant antibodies. Here we report an extension of this method referred to as fluorescent labeling in semi-solid medium (FLSSM) to detect recombinant proteins significantly smaller than antibodies, such as IGF-E5, a 25 kDa insulin-like growth factor derivative.     

Hapten-mediated immunopurification of membrane proteins labeled with fluorescein derivatives

The behavior of cell surface components labeled with fluorochromes can be studied by fluorescence microscopy and spectroscopy; further structural analyses would be facilitated by purification of the labeled components. We have developed a protocol for identifying the targets for labeling with fluorescein derivatives, by using 125I- diiodofluorescein isothiocyanate ( 125IFC ) and for isolating the labeled components with anti-IFC immunoadsorbents. Anti-IFC antibodies obtained from rabbits immunized with IFC-hemocyanin were purified by affinity chromatography and coupled to CNBr-activated Sepharose 4B. The anti-IFC immunoadsorbents could then be used to isolate the entire set of 125IFC -proteins from crude detergent extracts of labeled sea urchin sperm, with a 70% yield and a purification of more than 250 fold. Nonspecific binding of unlabeled proteins to the immunoadsorbent was insignificant. When the immunoadsorbent IFC-protein complex was used directly as an immunogen, antibodies were obtained that reacted with the underivatized proteins that were targets for IFC labeling, as indicated by immunoblotting after gel electrophoresis. The antibodies also reacted with the surface of unlabeled sperm as shown by immunofluorescence. Thus, by treating the IFC-sperm proteins as a class, we obtained antibodies that recognized the unlabeled proteins in situ or in cell extracts. This approach should be generally useful in obtaining reagents directed against specific cell surface components.     

通过荧光标记的凝胶阻滞技术分析Opaque2蛋白与ZmGRAS11启动子的结合位点

凝胶阻滞实验（electrophoretic mobility shift assay，EMSA）是研究蛋白质与核酸结合的一种关键实验技术。EMSA技术兴起以来，使用放射性同位素、生物素标记核酸探针的手段已经非常成熟，但这两种传统的标记技术分别具有放射性探针稳定性差和生物素检测步骤复杂等缺点。近年来，尽管荧光标记探针逐渐被应用于EMSA中，但是对于利用荧光标记探针的EMSA仍缺乏系统的报道。对荧光标记的EMSA技术流程进行了优化和系统总结；利用6-羧基荧光素（6-carboxy-fluoroscine，FAM）标记ZmGRAS11启动子探针，通过EMSA检测其与Opaque2蛋白的结合，明确了蛋白和探针的适宜比例为8∶1。对GCN4 motif序列碱基进行突变并利用EMSA分析Opaque2与ZmGRAS11启动子之间的结合位点，结果表明GCN4 motif的“TGAC”核心基序在ZmGRAS11启动子与Opaque2蛋白的结合中可能起到了关键作用。研究结果为进一步探究Opaque2-ZmGRAS11转录调控模块在玉米籽粒发育中的作用机理提供了数据支撑。     

Specific Lectin Binding to Beta1 Integrin and Fibronectin on the Apical Membrane of Madin-Darby Canine Kidney Cells

Although lectins have previously been used to identify specific cell types in the kidney and various other tissues, the proteins labeled were not identified. We hypothesized that fluorescently labeled lectins could provide a useful tool for direct labeling of membrane-associated glycoproteins. Protein fractions from Madin-Darby canine kidney (MDCK) cells were exposed to a panel of 16 fluorescently labeled lectins to identify suitable lectin-protein pairs. Peanut agglutinin (PNA) selectively bound a 220-240 kDa O-linked glycoprotein with a slightly acidic isoelectric point, while Sambucus nigra agglutinin (SNA) labeled a 130 kDa glycoprotein with a highly acidic isoelectric point. Both proteins were readily labeled by lectins applied to the apical surface of living confluent cells. The proteins were isolated by lectin affinity columns and identified by mass spectrometry. Peptides from the PNA-binding protein shared molecular weight and amino acid composition with fibronectin. Fragments of the SNA-binding protein showed amino-acid identity with peptides from beta1 integrin. The identities of these proteins were validated by Western blotting. Binding of PNA to a 220 kDa protein was inhibited by an anti-fibronectin antibody, and binding of a 130 kDa protein by SNA was diminished by an anti-beta1 integrin antibody. We conclude that PNA and SNA can be used as specific markers for fibronectin and beta1 integrin, respectively, in MDCK cells.     

A multiple high-resolution mini two-dimensional polyacrylamide gel electrophoresis system: imaging two-dimensional gels using a cooled charge-coupled device after staining with silver or labeling with fluorophore.

A multiple mini two-dimensional electrophoretic method which results in three two-dimensional protein spot patterns being positioned side by side in an individual gel has been developed. Preparation time has been minimized by employing disposable capillary tubes for the isoelectric focusing gels and reducing the number of second-dimensional gels required. Commercially available vertical slab units were used for the second-dimensional electrophoresis. The protein spot patterns were visualized either by staining the second-dimensional gel with silver or fluorescently labeling the focused proteins while present in the isoelectric focusing gel and subsequently electrophoresing them into the second-dimensional gel. The fluorescently labeled second-dimensional gel was imaged while still present in the glass mold immediately following electrophoresis. Two fluorophores were compared: 2-methoxy-2,4-diphenyl-3(2H)-furanone and 5-(4,6-dichlorotriazin-2-yl)aminofluorescein hydrochloride. A rapid imaging system based on a cooled charge-coupled device was used to view both the silver-stained and fluorescently labeled two-dimensional spot patterns. The sensitivity of detection of protein spots in the mini two-dimensional gels was similar for the two types of fluorescently labeled gels and the silver-stained gels.     

In vitro protein microarrays for detecting protein-protein interactions: application of a new method for fluorescence labeling of proteins

Protein microarrays or proteome chips are potentially powerful tools for comprehensive analysis of protein-protein interactions. In interaction analysis, a set of immobilized proteins is arrayed on slides and each slide is probed with a set of fluorescently labeled proteins. Here we have developed and tested an in vitro protein microarray, in which both arraying and probing proteins were prepared by cell-free translation. The in vitro synthesis of fluorescently labeled proteins was accomplished by a new method: a fluorophore-puromycin conjugate was incorporated into a protein at the C-terminus on the ribosome. The resulting fluorescently labeled proteins were confirmed to be useful for probing protein-protein interactions on protein microarrays in model experiments. Since the in vitro protein microarrays can easily be extended to a high-throughput format and also combined with in vitro display technologies such as the streptavidin-biotin linkage in emulsions method (Doi and Yanagawa, FEBS Lett. 1999, 457, 227-230), our method should be useful for large-scale analysis of protein-protein interactions.     

Molecular templates for bio-specific recognition by low-energy electron beam lithography

Protein patterning has become an important topic as advances are made in biologically integrated devices and protein chip technology. Versatile and effective patterning requires substrates that can be quantified, with active presentation of proteins and control over protein density and orientation. Herein we describe a model system and the use of low-energy electron beam lithography to pattern molecular templates for immobilization of antibodies through ligand recognition. The templates were patterned over a background of poly(ethylene glycol) (PEG) modified silicon oxide (SiO _x ). These substrates were exposed to a low-voltage (2 keV) electron beam to remove PEG selectively from exposed regions. These regions were then functionalized with a dinitrophenyl (DNP) ligand and tested for specific binding of fluorescently labeled anti-DNP antibodies. The PEG modified regions in conjunction with ligand-presenting regions in the patterned arrays substantially reduces non-specific adsorption of proteins, yielding a specific/nonspecific ratio of approx 10. The surface coverage of the biologically active DNP groups on SiO _x and the amount of immobilized antibody on DNP were measured with a fluorescence-based, enzyme-linked immunosorbent assay. The specificity of the interaction between DNP ligand and fluorescently labeled anti-DNP antibodies was evaluated with fluorescence microscopy. This approach to patterning of molecular templates and assays for quantification are generally applicable to immobilization of any ligand-receptor pair on a wide range of substrates.     

Neuron-specific membrane glycoproteins promoting neurite fasciculation in Aplysia californica

We have generated a library of mouse monoclonal antibodies against membrane proteins of the nervous system of the marine snail Aplysia californica. Two of these antibodies, 4E8 and 3D9, recognize a group of membrane glycoproteins with molecular masses of 100-150 kD. We have called these proteins ap100, from the molecular mass of the most abundant species. Based on Western blots, these proteins appear to be specific for the nervous system. They are enriched in the neuropil of central nervous system ganglia, and are present on the surface of neurites and growth cones of neurons in culture. They are not expressed on the surface of nonneuronal cells. Staining of living cells with fluorescently labeled mAb demonstrates that the epitope(s) are on the outside of the cell. The antibodies against the proteins defasciculate growing axons and alter the morphology of growth cones, but affect much less adhesion between neuritic shafts. In addition, the level of expression of these molecules appears to correlate with the degree of fasciculation of neurites. These observations suggest that the ap100 proteins are cell adhesion molecules that play a role in axon growth in the nervous system of Aplysia. The fact that they are enriched in the neuropil and possibly in varicosities suggest that they may also be relevant for the structure of mature synapses.     

Polyethyleneimine as a transmembrane carrier of fluorescently labeled proteins and antibodies

Polyethyleneimine (PEI) has been used previously as a nonviral DNA transfer vector. In this article, we demonstrate its use as a vehicle for transmembrane delivery of proteins in cell culture conditions. Linking proteins to PEI required no other treatment beyond mixing them with PEI. The bond between PEI and protein combined at optimal ratios was maintained in electrophoresis, even in the presence of 2.5% sodium dodecyl sulfate (SDS). The optimal time for delivery of proteins was determined to be 24 h. We have successfully delivered an Alexa 488-labeled avidin protein into human glioblastoma cells. A functional antibody against the nuclear protein lamin was delivered into human fibroblasts and reacted with lamin inside live cells. PEI-based delivery of antibodies and fluorescently labeled proteins can be used for fluorescent detection, tracking, and evaluation of cellular protein function in vivo.     

Single-molecule analysis of DNA replication in Xenopus egg extracts

The recent advent in single-molecule imaging and manipulation methods has made a significant impact on the understanding of molecular mechanisms underlying many essential cellular processes. Single-molecule techniques such as electron microscopy and DNA fiber assays have been employed to study the duplication of genome in eukaryotes. Here, we describe a single-molecule assay that allows replication of DNA attached to the functionalized surface of a microfluidic flow cell in a soluble Xenopus leavis egg extract replication system and subsequent visualization of replication products via fluorescence microscopy. We also explain a method for detection of replication proteins, through fluorescently labeled antibodies, on partially replicated DNA immobilized at both ends to the surface.     

Immunolocalization of the vacuolar-type (H+)-ATPase from clathrin-coated vesicles.

Proton-translocating ATPases of the vacuolar class (V-ATPases) are found in a variety of animal cell compartments that participate in vesicular membrane transport, including clathrin-coated vesicles, endosomes, the Golgi apparatus, and lysosomes. The exact structural relationship that exists among the V-ATPases of these intracellular compartments is not currently known. In the present study, we have localized the V-ATPase by light and electron microscopy, using monoclonal antibodies that recognize the V-ATPase present in clathrin-coated vesicles. Localization using light microscopy and fluorescently labeled antibodies reveals that the V-ATPase is concentrated in the juxtanuclear region, where extensive colocalization with the Golgi marker wheat germ agglutinin is observed. The V-ATPase is also present in approximately 60% of endosomes and lysosomes fluorescently labeled using alpha 2-macroglobulin as a marker for the receptor-mediated endocytic pathway. Localization using transmission electron microscopy and colloidal gold-labeled antibodies reveals that the V-ATPase is present at and near the plasma membrane, alone or in association with clathrin. These results provide evidence that the V-ATPases of plasma membrane, endosomes, lysosomes, and the Golgi apparatus are immunologically related to the V-ATPase of clathrin-coated vesicles.     

A method for detecting proteins immobilized on nitrocellulose membranes by in situ derivatization with fluorescein isothiocyanate

A method for the fluorescent staining of proteins on nitrocellulose filters is described. The single step procedure uses a 100 microgram/ml solution of fluorescein isothiocyanate in sodium carbonate buffer, pH 9.5. The proteins are visible under uv light within 30 s and the staining reaction is virtually complete after 10 min. The method can detect a minimum of 50 ng protein/band providing a sensitivity similar to that obtained with anionic dye stains. The method is suitable for blots prepared from both isoelectric focusing gels and sodium dodecyl sulfate-polyacrylamide gels. The fluorescently labeled proteins can be probed using immunochemical techniques with the retention of fluorescence. The method can therefore be used to accurately locate antigens among a number of proteins and allows the sensitive and rapid detection of marker proteins directly on the blot.     

Anti-KDEL-coated nanoparticles: A promising tumor targeting approach for ovarian cancer?

The purpose of this study was to target ovarian cancer cells by coupling paclitaxel (Tx)-loaded nanoparticles (NPs-Tx) to antibodies against KDEL sequence, able to recognize GRP94 and GRP78 that are located at cell surface in cancer cells whereas they are in the endoplasmic reticulum in healthy cells. Tx-loaded poly (dl-lactic acid) nanoparticles coated with anti-KDEL antibodies (NPs-Tx-KDEL) were successfully prepared and characterized. Interaction between tumor cells and NPs-Tx or NPs-Tx-KDEL was observed by microscopy with fluorescently labeled NPs and the efficacy of the different formulations was compared by a viability assay.     

Following the Fate In Vivo of COPI Vesicles Generated In Vitro

COPI vesicles are a class of transport carriers that function in the early secretory pathway. Their fate and function are still controversial. This includes their contribution to bidirectional transport within the Golgi apparatus and their role during cell division. Here we describe a method that should address several open questions about the fate and function of COPI vesicles in vivo . To this end, fluorescently labeled COPI vesicles were generated in vitro from isolated rat liver Golgi membranes, labeled with the fluorescent dyes Alexa-488 or Alexa-568. These vesicles appeared to be active and colocalized with endogenous Golgi membranes within 30 min after microinjection into mammalian cells. The COPI vesicle-derived labeled membrane proteins could be classified into two types that behaved like endogenous proteins after Brefeldin A treatment.     

One-pot fluorescent labeling of xyloglucan oligosaccharides with sulforhodamine

Xyloglucan oligosaccharides fluorescently labeled with sulforhodamine have proved to be a valuable tool in the assessment of transglycosylating activity of plant xyloglucan endotransglucosylase/hydrolase (XTH; EC 2.4.1.207). Here we describe a simple and fast procedure for their preparation. Accordingly, the starting xyloglucan-derived oligosaccharides are in the first step converted to their corresponding 1-amino-1-deoxyalditols (glycamines) by incubation with ammonium acetate and NaCNBH(3) at 80 degrees C for 2-4 h, and in the second step, the glycamines are reacted with Lissamine rhodamine B sulfonyl chloride to obtain fluorescently labeled derivatives of the oligosaccharide glycamines. All operations are carried out in a single centrifuge tube and the products from the individual reaction steps are isolated on the basis of their differential solubility in organic solvents. Using the described protocol, the whole procedure can be accomplished in less than 24 h. The sulforhodamine-labeled xyloglucan oligosaccharides thus obtained proved suitable as substrates for a sensitive fluorescence assay of the transglycosylating activity of XTH.     

Increasing the sensitivity of reverse phase protein arrays by antibody-mediated signal amplification

Background  

Reverse phase protein arrays (RPPA) emerged as a useful experimental platform to analyze biological samples in a high-throughput format. Different signal detection methods have been described to generate a quantitative readout on RPPA including the use of fluorescently labeled antibodies. Increasing the sensitivity of RPPA approaches is important since many signaling proteins or posttranslational modifications are present at a low level.