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.     

Simultaneous trichromatic fluorescence detection of proteins on Western blots using an amine-reactive dye in combination with alkaline phosphatase- and horseradish peroxidase-antibody conjugates

Three-color fluorescence detection methods are described based upon covalently coupling the dye 2-methoxy-2,4-diphenyl-2(2H)-furanone (MDPF) to proteins immobilized on poly(vinylidene difluoride) (PVDF) membranes, followed by detection of target proteins using alkaline-phosphatase-conjugated reporter molecules in combination with the fluorogenic substrate 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) phosphate (DDAO-phosphate) as well as horseradish peroxidase-conjugated reporter molecules in combination with the new fluorogenic substrate Amplex Gold reagent. This results in all proteins in the profile being visualized as fluorescent blue signal, those detected specifically with the alkaline phosphatase conjugate appearing as fluorescent red signal and those detected specifically with the horseradish peroxidase conjugate appearing as fluorescent yellow signal. Using conventional secondary antibodies, two different targets may be identified as long as primary antibodies generated from two different species are used in the analysis. However, Zenon antibody labeling technology eliminates this restriction, permitting the simultaneous use of two different mouse monoclonal antibodies or two different rabbit polyclonal antibodies in the same electroblotting experiment. The trichromatic detection system is broadly compatible with UV epi-illuminators combined with photographic or charge-coupled device (CCD) cameras, and xenon-arc sources equipped with appropriate excitation/emission filters. Alternatively, the enzyme conjugates may be detected using a laser-based gel scanner. The trichromatic method permits detection of low nanogram amounts of protein and allows for unambiguous identification of two different target proteins relative to the entire protein profile on a single electroblot, precluding any requirement for running replicate gels that would otherwise require separate visualization of total proteins and subsequent alignment with multiple chemiluminescent or colorimetric signals generated on different electroblots.     

Use of green fluorescent protein color variants expressed on stable broad-host-range vectors to visualize rhizobia interacting with plants

We developed two sets of broad-host-range vectors that drive expression of the green fluorescent protein (GFP) or color variants thereof (henceforth collectively called autofluorescent proteins [AFPs]) from the lac promoter. These two sets are based on different replicons that are maintained in a stable fashion in Escherichia coli and rhizobia. Using specific filter sets or a dedicated confocal laser scanning microscope setup in which emitted light is split into its color components through a prism, we were able to unambiguously identify bacteria expressing enhanced cyan fluorescent protein (ECFP) or enhanced yellow fluorescent protein (EYFP) in mixtures of the two. Clearly, these vectors will be valuable tools for competition, cohabitation, and rescue studies and will also allow the visualization of interactions between genetically marked bacteria in vivo. Here, we used these vectors to visualize the interaction between rhizobia and plants. Specifically, we found that progeny from different rhizobia can be found in the same nodule or even in the same infection thread. We also visualized movements of bacteroids within plant nodule cells.     

A blue dive: from ‘blue fingers’ to ‘blue silver’. A comparative overview of staining methods for in-gel proteomics

Gel-based proteomics are the most useful method for protein separation, even when compared with gel-free proteomics. Proteomic analysis by 2D gel electrophoresis (2-DE) with immobilized pH gradients is in turn the best approach to large-scale protein-expression screening. Spots visualization is pivotal for protein identification by mass spectrometry. Commonly used staining methods with excellent mass spectrometry compatibility are coomassie brilliant blue (CBB) or fluorescent dyes. In this study, an implementation of ‘blue silver’ colloidal CBB staining, characterized by high sensitivity and immediate low background, is discussed. The sensitivity of classical, colloidal and ‘blue silver’ CBB staining methods was compared on monodimensional and 2-DE gels. The implementation of the ‘blue silver’ method performs better, provided the physical state of the micelles is respected. An example of a 2-DE of human urine treated with combinatorial peptide ligand libraries demonstrates that implemented ‘blue silver’ can evidence the complexity of the sample.     

Improved tree-ring visualization using autofluorescence

The great diversity of wood anatomical features found in trees worldwide results in a broad variety of growth-ring boundary types that are not always easy to recognize, especially in tropical woods. However, the presence of clearly visible limits between tree rings is essential for any tree-ring studies. Here, we propose the use of autofluorescence of wood in order to enhance tree-ring visualization. The multispectral light emitted from the fluorescence stereomicroscope can be filtered in specific wavelengths to improve the visualization of wood anatomical features. To evaluate the effectiveness of this technique, we compared visualization under natural light, GFP (green fluorescent protein) filter, RFP (red fluorescent protein) filter and UV filter. We tested this technique with a set of 38 tree species with different types of growth-ring boundaries. Although results are species-specific, fluorescence has been shown to improve the visualization of growth-ring boundaries by enhancing the contrast among cell types. It may highlight fibrous zones (e.g. Cavanillesia arborea, Aspidosperma polyneuron), different porosity patterns (e.g. Myracrodruon urundeuva), secretory canals (e.g. Copaifera langsdorffii), and parenchyma bands (e.g. Tipuana tipu). Fluorescence allows the visualization of growth-ring boundaries in species that were previously described as having indistinct growth rings under natural light. For species with clear tree-ring boundaries such as Cedrela fissilis and Hymenaea courbaril, this approach aids the identification of false rings. In addition to the visualization of growth-ring boundaries, autofluorescence may be useful for other qualitative and quantitative analyses of wood anatomy, such as wood identification and automated measurements of anatomical features. Scientists struggling with tree-ring counting and cross-dating due to difficult tree-ring visualization may find fluorescence useful. It may also aid to identify new species suitable for tree-ring studies.     

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.     

Intrinsic Protein Fluorescence Interferes with Detection of Tear Glycoproteins in SDS-Polyacrylamide Gels Using Extrinsic Fluorescent Dyes

Intrinsic protein fluorescence may interfere with the visualization of proteins after SDS-polyacrylamide electrophoresis. In an attempt to analyze tear glycoproteins in gels, we ran tear samples and stained the proteins with a glycoprotein-specific fluorescent dye. The fluorescence detected was not limited to glycoproteins. There was strong intrinsic fluorescence of proteins normally found in tears after soaking the gels in 40% methanol plus 1-10% acetic acid and, to a lesser extent, in methanol or acetic acid alone. Nanograms of proteins gave visible native fluorescence and interfere with extrinsic fluorescent dye detection. Poly-L-lysine, which does not contain intrinsically fluorescent amino acids, did not fluoresce.     

Ultraviolet shadowing nucleic acids on nylon membranes

We describe a method for the direct visualization of nucleic acids on nylon membranes. Nylon is weakly fluorescent under short wave ultraviolet light allowing membrane-bound nucleic acids to be detected with a sensitivity of 10 ng. This procedure involves no staining or destaining of the gels prior to transfer, does not require duplicate sample lanes or blots, and does not interfere with transfer of the nucleic acid to the membrane or subsequent hybridization.     

Vibrio azureus emits blue-shifted light via an accessory blue fluorescent protein

Luminous marine bacteria usually emit bluish-green light with a peak emission wavelength (λ(max) ) at about 490?nm. Some species belonging to the genus Photobacterium are exceptions, producing an accessory blue fluorescent protein (lumazine protein: LumP) that causes a blue shift, from λ(max) ≈?490 to λ(max) ≈?476?nm. However, the incidence of blue-shifted light emission or the presence of accessory fluorescent proteins in bacteria of the genus Vibrio has never been reported. From our spectral analysis of light emitted by 16 luminous strains of the genus Vibrio, it was revealed that most strains of Vibrio azureus emit a blue-shifted light with a peak at approximately 472?nm, whereas other Vibrio strains emit light with a peak at around 482?nm. Therefore, we investigated the mechanism underlying this blue shift in V.?azureus NBRC 104587(T) . Here, we describe the blue-shifted light emission spectra and the isolation of a blue fluorescent protein. Intracellular protein analyses showed that this strain had a blue fluorescent protein (that we termed VA-BFP), the fluorescent spectrum of which was almost identical to that of the in vivo light emission spectrum of the strain. This result strongly suggested that VA-BFP was responsible for the blue-shifted light emission of V.?azureus.     

Selective photolabeling of proteins using photoactivatable GFP

Today's cell biologists rely on an assortment of advances in microscopy methods to study the inner workings of cells and tissues. Among these advances are fluorescent proteins which can be used to tag specifically and, in many cases, non-invasively proteins of interest within a living cell. Introduction of DNA encoding the fluorescently tagged protein of interest into a cell readily allows the visualization of the protein's localization and time-lapse imaging allows the movement of the structure or organelle to which the protein is localized to be observed. To monitor the movement of the protein within the population, researchers generally have to highlight a pool of molecules by perturbing the steady-state fluorescence. This perturbation has traditionally been performed by photobleaching the molecules within a selected region of the cell and monitoring the recovery of molecules into this region or the loss of molecules within other regions. Fluorescent proteins are now available, which allow a pool of molecules to be highlighted directly by photoactivation. Here, we discuss the technical aspects for using one of these recently developed photoactivatable fluorescent proteins, PA-GFP.     

Selective silver-staining methods for RNA and proteins in the same polyacrylamide gels

Two silver-staining methods for selective and ultrasensitive detection of RNAs and proteins in the same polyacrylamide gels were developed, both derived from procedures recommended for protein staining. The first, a double-staining technic with Coomassie brilliant blue and ammoniacal silver, allows visualization of RNAs as negative bands and proteins as dark brown bands. The second is also a double-staining technique, but uses silver in both steps. This second method develops the RNA bands first and then the protein bands. These techniques, especially the second, permit characterization of the different components of ribonucleoproteic complexes in the same electrophoresis gels.     

Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate goal is to image neuronal activity in vivo, one must be able to image activity of a single cell to ensure successful in vivo preparations. This procedure will describe how to image membrane potential in a single cell to provide a foundation to eventually image in vivo. Here we describe methods for imaging GEVIs consisting of a voltage-sensing domain fused to either a single fluorescent protein (FP) or two fluorescent proteins capable of Förster resonance energy transfer (FRET) in vitro. Using an image splitter enables the projection of images created by two different wavelengths onto the same charge-coupled device (CCD) camera simultaneously. The image splitter positions a second filter cube in the light path. This second filter cube consists of a dichroic and two emission filters to separate the donor and acceptor fluorescent wavelengths depending on the FPs of the GEVI. This setup enables the simultaneous recording of both the acceptor and donor fluorescent partners while the membrane potential is manipulated via whole cell patch clamp configuration. When using a GEVI consisting of a single FP, the second filter cube can be removed allowing the mirrors in the image splitter to project a single image onto the CCD camera.     

Simultaneous red/green dual fluorescence detection on electroblots using BODIPY TR-X succinimidyl ester and ELF 39 phosphate

A two-color fluorescence detection method is described based upon covalently coupling the succinimidyl ester of BODIPY TR-X dye to proteins immobilized on polyvinylidene difluoride membranes, followed by detection of target proteins using the fluorogenic, precipitating substrate ELF 39-phosphate in combination with alkaline phosphatase conjugated reporter molecules. This results in all proteins in the profile being visualized as fluorescent red signal while those detected specifically with the alkaline phosphatase conjugate appear as fluorescent green signal. The dichromatic detection system is broadly compatible with ultraviolet epi- or trans-illuminators combined with photographic or charge-coupled device cameras, and xenon-arc sources equipped with appropriate excitation/emission filters. The dichromatic 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. Combining the detection approach with an Alexa Fluor 350 dye conjugated monoclonal antibody permits simultaneous fluorescence detection of two antigens and the total protein profile on the same electroblot.     

Macular pigments lutein and zeaxanthin as blue light filters studied in liposomes.

Lutein and zeaxanthin are the predominant carotenoids in the human macula lutea. Epidemiological data suggest that an increased intake of a lutein-rich diet correlates with a diminished risk for age-related macular degeneration, a major cause of impaired vision in the elderly. Filtering of blue light has been proposed as a possible mechanism of protection. Here, the blue light filter efficacy of carotenoids was investigated in unilamellar liposomes loaded in the hydrophilic core space with a fluorescent dye, Lucifer yellow, excitable by blue light. Carotenoids were incorporated into the lipophilic membrane. Fluorescence emission in carotenoid-containing liposomes was lower than in carotenoid-free controls when exposed to blue light, indicating a filter effect. Filter efficacy was in the order lutein > zeaxanthin > beta-carotene > lycopene. Some of the difference in blue light filter efficacy of carotenoids is attributable to differences in extinction coefficients, and a major further contribution is suggested to be related to the orientation of the incorporated molecules in the liposomal membrane.     

Characterization of Fluorescent Proteins for Three- and Four-Color Live-Cell Imaging in S. cerevisiae

Saccharomyces cerevisiae are widely used for imaging fluorescently tagged protein fusions. Fluorescent proteins can easily be inserted into yeast genes at their chromosomal locus, by homologous recombination, for expression of tagged proteins at endogenous levels. This is especially useful for incorporation of multiple fluorescent protein fusions into a single strain, which can be challenging in organisms where genetic manipulation is more complex. However, the availability of optimal fluorescent protein combinations for 3-color imaging is limited. Here, we have characterized a combination of fluorescent proteins, mTFP1/mCitrine/mCherry for multicolor live cell imaging in S. cerevisiae. This combination can be used with conventional blue dyes, such as DAPI, for potential four-color live cell imaging.     

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.     

1.8 A bright-state structure of the reversibly switchable fluorescent protein Dronpa guides the generation of fast switching variants

RSFPs (reversibly switchable fluorescent proteins) may be repeatedly converted between a fluorescent and a non-fluorescent state by irradiation and have attracted widespread interest for many new applications. The RSFP Dronpa may be switched with blue light from a fluorescent state into a non-fluorescent state, and back again with UV light. To obtain insight into the underlying molecular mechanism of this switching, we have determined the crystal structure of the fluorescent equilibrium state of Dronpa. Its bicyclic chromophore is formed spontaneously from the Cys62-Tyr63-Gly64 tripeptide. In the fluorescent state, it adopts a slightly non-coplanar cis conformation within the interior of a typical GFP (green fluorescent protein) b-can fold. Dronpa shares some structural features with asFP595, another RSFP whose chromophore has previously been demonstrated to undergo a cis-trans isomerization upon photoswitching. Based on the structural comparison with asFP595, we have generated new Dronpa variants with an up to more than 1000-fold accelerated switching behaviour. The mutations which were introduced at position Val157 or Met159 apparently reduce the steric hindrance for a cis-trans isomerization of the chromophore, thus lowering the energy barrier for the blue light-driven on-to-off transition. The findings reported in the present study support the view that a cis-trans isomerization is one of the key events common to the switching mechanism in RSFPs.     

Controlled three-dimensional immobilization of biomolecules on chemically patterned surfaces

We used electron-beam lithography to fabricate chemical nanostructures, i.e. amino groups in aromatic self-assembled monolayers (SAMs) on gold surfaces. The amino groups are utilized as reactive species for mild covalent attachment of fluorescently labeled proteins. Since non-radiative energy transfer results in strong quenching of fluorescent dyes in the vicinity of the metal surfaces, different labeling strategies were investigated. Spacers of varying length were introduced between the gold surface and the fluorescently labeled proteins. First, streptavidin was directly coupled to the amino groups of the SAMs via a glutaraldehyde linker and fluorescently labeled biotin (X-Biotin) was added, resulting in a distance of approximately 2 nm between the dyes and the surface. Scanning confocal fluorescence images show that efficient energy transfer from the dye to the surface occurs, which is reflected in poor signal-to-background (S/B) ratios of approximately 1. Coupling of a second streptavidin layer increases the S/B-ratio only slightly to approximately 2. The S/B-ratio of the fluorescence signals could be further increased to approximately 4 by coupling of an additional fluorescently labeled antibody layer. Finally, we introduced tetraethylenepentamine as functional spacer molecule to diminish fluorescence quenching by the surface. We demonstrate that the use of this spacer in combination with multiple antibody layers enables the controlled fabrication of highly fluorescent three-dimensional nanostructures with S/B-ratios of >20. The presented technique might be used advantageously for the controlled three-dimensional immobilization of single protein or DNA molecules and the well-defined assembly of protein complexes.     

The hazards of DAPI photoconversion: effects of dye, mounting media and fixative, and how to minimize the problem

Immunocytochemistry is a powerful tool for detection and visualization of specific molecules in living or fixed cells, their localization and their relative abundance. One of the most commonly used fluorescent DNA dyes in immunocytochemistry applications is 4′,6-diamidino-2-phenylindole dihydrochloride, known as DAPI. DAPI binds strongly to DNA and is used extensively for visualizing cell nuclei. It is excited by UV light and emits characteristic blue fluorescence. Here, we report a phenomenon based on an apparent photoconversion of DAPI that results in detection of a DAPI signal using a standard filter set for detection of green emission due to blue excitation. When a sample stained with DAPI only was first imaged with the green filter set (FITC/GFP), only a weak cytoplasmic autofluorescence was observed. Next, we imaged the sample with a DAPI filter set, obtaining a strong nuclear DAPI signal as expected. Upon reimaging the same samples with a FITC/GFP filter set, robust nuclear fluorescence was observed. We conclude that excitation with UV results in a photoconversion of DAPI that leads to detection of DAPI due to excitation and emission in the FITC/GFP channel. This phenomenon can affect data interpretation and lead to false-positive results when used together with fluorochrome-labeled nuclear proteins detected with blue excitation and green emission. In order to avoid misinterpretations, extra precaution should be taken to prepare staining solutions with low DAPI concentration and DAPI (UV excitation) images should be acquired after all other higher wavelength images. Of various DNA dyes tested, Hoechst 33342 exhibited the lowest photoconversion while that for DAPI and Hoechst 33258 was much stronger. Different fixation methods did not substantially affect the strength of photoconversion. We also suggest avoiding the use of mounting medium with high glycerol concentrations since glycerol showed the strongest impact on photoconversion. This photoconversion effect cannot be avoided even when using narrow bandpass filter sets.