Photochemical labeling of membrane hydrophobic core of human erythrocytes using a new photoactivable reagent 2-[3H]diazofluorene

Photoactivable reagents have been useful for studying the structural aspects of membrane hydrophobic core. We have reported earlier (Anjaneyulu, P.S.R., and Lala, A. K. (1982) FEBS Lett. 146, 165-167) the use of diazofluorene as a probe for fluorescent photochemical labeling of hydrophobic core in artificial membranes. To quantitate and enhance the monitoring ability of this probe, we have synthesized 2-[3H]diazofluorene of high specific activity. This reagent rapidly partitions into phosphatidylcholine vesicles and selectively labels the fatty acyl chains of phosphatidylcholine. The insertion yield (13%) is not affected by the presence of scavengers like reduced glutathione. 2-[3H]Diazofluorene also readily partitions into erythrocyte membranes and on photolysis labels the membrane. The overall insertion was 48% with 9.7% in protein fraction and the rest in lipids. The distribution of radioactivity in labeled protein fraction was restricted to integral membrane proteins with Band 3 being the major protein labeled. There is little or no labeling associated with extrinsic proteins like spectrin. Further analysis of labeled Band 3 by treatment with chymotrypsin indicated that the labeling was restricted to the membrane spanning CH-17 and CH-35 fragments. No labeling of the cytoplasmic fragment of Band 3 could be observed. 2-[3H]Diazofluorene should prove useful for studying integral membrane proteins and their membrane-spanning regions.     

Rational design of a monomeric and photostable far‐red fluorescent protein for fluorescence imaging in vivo

Fluorescent proteins (FPs) are powerful tools for cell and molecular biology. Here based on structural analysis, a blue‐shifted mutant of a recently engineered monomeric infrared fluorescent protein (mIFP) has been rationally designed. This variant, named iBlueberry, bears a single mutation that shifts both excitation and emission spectra by approximately 40 nm. Furthermore, iBlueberry is four times more photostable than mIFP, rendering it more advantageous for imaging protein dynamics. By tagging iBlueberry to centrin, it has been demonstrated that the fusion protein labels the centrosome in the developing zebrafish embryo. Together with GFP‐labeled nucleus and tdTomato‐labeled plasma membrane, time‐lapse imaging to visualize the dynamics of centrosomes in radial glia neural progenitors in the intact zebrafish brain has been demonstrated. It is further shown that iBlueberry can be used together with mIFP in two‐color protein labeling in living cells and in two‐color tumor labeling in mice.     

Selective chemical labeling of proteins in living cells

Labeling proteins with fluorophores, affinity labels or other chemically or optically active species is immensely useful for studying protein function in living cells or tissue. The use of genetically encoded green fluorescent protein and its variants has been particularly valuable in this regard. In an effort to increase the diversity of available protein labels, various efforts to append small molecules to selected proteins in vivo have been reported. This review discusses recent advances in selective, in vivo protein labeling based on small molecule ligand-receptor interactions, intein-mediated processes, and enzyme-catalyzed protein modifications.     

Multiple labeling of antibodies with dye/DNA conjugate for sensitivity improvement in fluorescence immunoassay

Fluorescence immunoassays are widely used in life science research, medical diagnostics, and environmental monitoring due to the intrinsically high specificity, simplicity, and versatility of immunoassays, as well as the availability of a large variety of fluorescent labeling molecules. However, the sensitivity needs to be improved to meet the ever-increasing demand in the new proteomics era. Here, we report a simple method of attaching multiple fluorescent labels on an antibody with a dye/DNA conjugate to increase the immunoassay sensitivity. In the work, mouse IgG adsorbed on the surface of a 96-well plate was detected by its immunoreaction with biotinylated goat anti-mouse antibody. A 30 base pair double-stranded oligonucleotide terminated with biotin was attached to the antibody through the biotin/streptavidin/biotin interaction. Multiple labeling of the antibody was achieved after a fluorescent DNA probe was added into the solution and bound to the oligonucleotide at high ratios. By comparison with fluorescein-labeled streptavidin, the assay with the dye/DNA label produced up to 10-fold increase in fluorescence intensity, and consequently about 10-fold lower detection limit. The multiple labeling method uses readily available reagents, and is simple to implement. Further sensitivity improvement can be obtained by using longer DNAs for antibody labeling, which can incorporate more fluorescent dyes on each DNA.     

Split-wrmScarlet and split-sfGFP: tools for faster,easier fluorescent labeling of endogenous proteins in Caenorhabditis elegans

We create and share a new red fluorophore, along with a set of strains, reagents and protocols, to make it faster and easier to label endogenous Caenorhabditis elegans proteins with fluorescent tags. CRISPR-mediated fluorescent labeling of C. elegans proteins is an invaluable tool, but it is much more difficult to insert fluorophore-size DNA segments than it is to make small gene edits. In principle, high-affinity asymmetrically split fluorescent proteins solve this problem in C. elegans: the small fragment can quickly and easily be fused to almost any protein of interest, and can be detected wherever the large fragment is expressed and complemented. However, there is currently only one available strain stably expressing the large fragment of a split fluorescent protein, restricting this solution to a single tissue (the germline) in the highly autofluorescent green channel. No available C. elegans lines express unbound large fragments of split red fluorescent proteins, and even state-of-the-art split red fluorescent proteins are dim compared to the canonical split-sfGFP protein. In this study, we engineer a bright, high-affinity new split red fluorophore, split-wrmScarlet. We generate transgenic C. elegans lines to allow easy single-color labeling in muscle or germline cells and dual-color labeling in somatic cells. We also describe a novel expression strategy for the germline, where traditional expression strategies struggle. We validate these strains by targeting split-wrmScarlet to several genes whose products label distinct organelles, and we provide a protocol for easy, cloning-free CRISPR/Cas9 editing. As the collection of split-FP strains for labeling in different tissues or organelles expands, we will post updates at doi.org/10.5281/zenodo.3993663     

Immunofluorescent labeling of cancer marker Her2 and other cellular targets with semiconductor quantum dots

Semiconductor quantum dots (QDs) are among the most promising emerging fluorescent labels for cellular imaging. However, it is unclear whether QDs, which are nanoparticles rather than small molecules, can specifically and effectively label molecular targets at a subcellular level. Here we have used QDs linked to immunoglobulin G (IgG) and streptavidin to label the breast cancer marker Her2 on the surface of fixed and live cancer cells, to stain actin and microtubule fibers in the cytoplasm, and to detect nuclear antigens inside the nucleus. All labeling signals are specific for the intended targets and are brighter and considerably more photostable than comparable organic dyes. Using QDs with different emission spectra conjugated to IgG and streptavidin, we simultaneously detected two cellular targets with one excitation wavelength. The results indicate that QD-based probes can be very effective in cellular imaging and offer substantial advantages over organic dyes in multiplex target detection.     

Fluorescent labeling and modification of proteins

This review provides an outline for fluorescent labeling of proteins. Fluorescent assays are very diverse providing the most sensitive and robust methods for observing biological processes. Here, different types of labels and methods of attachment are discussed in combination with their fluorescent properties. The advantages and disadvantages of these different methods are highlighted, allowing the careful selection for different applications, ranging from ensemble spectroscopy assays through to single-molecule measurements.     

Quantitative analysis of protein phosphorylation status and protein kinase activity on microarrays using a novel fluorescent phosphorylation sensor dye

Ultrasensitive detection of minute amounts of phosphorylated proteins and peptides is a key requirement for unraveling many of the most important signal transduction pathways in mammalian systems. Protein microarrays are potentially useful tools for sensitive screening of global protein expression and post-translational modifications, such as phosphorylation. However, the analysis of signaling pathways has been hampered by a lack of reagents capable of conveniently detecting the targets of protein kinases. Historically, phosphorylation detection methods have relied upon either radioisotopes ((gamma-(32)P)ATP(gamma-(33)P)ATP labeling) or phosphoamino acid-selective antibodies. Both of these methods suffer from relatively well-known shortcomings. In this study, a small molecule fluorophore phosphosensor technology is described, referred to as Pro-Q Diamond dye, which is capable of ultrasensitive global detection and quantitation of phosphorylated amino acid residues in peptides and proteins displayed on microarrays. The utility of the fluorescent Pro-Q Diamond phosphosensor dye technology is demonstrated using phosphoproteins and phosphopeptides as well as with protein kinase reactions performed in miniaturized microarray assay format. Instead of applying a phosphoamino acid-selective antibody labeled with a fluorescent or enzymatic tag for detection, a small, fluorescent probe is employed as a universal sensor of phosphorylation status. The detection limit for phosphoproteins on a variety of different commercially available protein array substrates was found to be 312-625 fg, depending upon the number of phosphate residues. Characterization of the enzymatic phosphorylation of immobilized peptide targets with Pro-Q Diamond dye readily permits differentiation between specific and non-specific peptide labeling at picogram to subpicogram levels of detection sensitivity.     

Mass spectrometric analysis of protein interactions

Mass spectrometry is a powerful tool for identification of interaction partners and structural characterization of protein interactions because of its high sensitivity, mass accuracy and tolerance towards sample heterogeneity. Several tools that allow studies of protein interaction are now available and recent developments that increase the confidence of studies of protein interaction by mass spectrometry include quantification of affinity-purified proteins by stable isotope labeling and reagents for surface topology studies that can be identified by mass-contributing reporters (e.g. isotope labels, cleavable cross-linkers or fragment ions. The use of mass spectrometers to study protein interactions using deuterium exchange and for analysis of intact protein complexes recently has progressed considerably.     

Cyanine dye labeling reagents containing isothiocyanate groups

New isothiocyanate derivatives of cyanine dyes were synthesized as fluorescent covalent labeling reagents for proteins and other biomolecules. These dyes have maximum absorbance in the red and near infrared regions of the spectrum, have high extinction coefficients and have adequate quantum yields. Incorporating two alkyl sulfonate groups in the dye structures increases their water solubility, which is beneficial for labeling biological molecules in aqueous solution. Reactivities of proteins with these new cyanines are similar to their reactivities with fluorescein isothiocyanate. These new labeling reagents are complementary to the fluorescein and rhodamine reagents, expanding the possibilities of multicolor analyses. Sheep anti-mouse-IgG antibody was labeled with a pentamethine cyanine dye (CY5.8-ITC) and used with a fluoresceinated antibody as a second reagent for detecting human T-cell subsets by flow cytometry.     

Ultrasensitive detection of biomolecules with fluorescent dye-doped nanoparticles

Fluorescent-labeled molecules have been used extensively for a wide range of applications in biological detection and diagnosis. A new form of highly luminescent and photostable nanoparticles was generated by doping the fluorescent dye tris(2'2-bipyridyl)dichlororuthenium(II)hexahydrate (Rubpy) inside silica material. Because thousands of fluorescent dye molecules are encapsulated in the silica matrix that also serves to protect Rubpy dye from photodamaging oxidation, the Rubpy-dye-doped nanoparticles are extremely bright and photostable. We have used these nanoparticles successfully in various fluorescence labeling techniques, including fluorescent-linked immunosorbent assay, immunocytochemistry, immunohistochemistry, DNA microarray, and protein microarray. By combining the high-intensity luminescent nanoparticles with the specificity of antibody-mediated recognition, ultrasensitive target detection has been achieved. In all cases, assay results clearly demonstrated the superiority of the nanoparticles over organic fluorescent dye molecules and quantum dots in probe labeling for sensitive target detection. These results demonstrate the potential to apply these newly developed fluorescent nanoparticles in various biodetection systems.     

Advances in the study of luminescence probes for proteins

Spectral probes (or labels) have been widely used for the investigation and determination of proteins and have made considerable progress. Traditional luminescence probes include fluorescent derivatizing reagents, fluorescent probes and chemiluminescence probes which continue to develop. Of them, near infrared (NIR) fluorescent probes are especially suitable for the determination of biomolecules including proteins, so their development has been rapid. Novel luminescence probes (such as nanoparticle probes and molecular beacons) and resonance light scattering probes recently appeared in the literature. Preliminary results indicate that they possess great potential for ultrasensitive protein detection. This review summarizes recent developments of the above-mentioned probes for proteins and 195 references are cited.     

A brilliant monomeric red fluorescent protein to visualize cytoskeleton dynamics in Dictyostelium

Red fluorescent proteins (RFPs) combined with GFP are attractive probes for double-fluorescence labeling of proteins in live cells. However, the application of these proteins is restrained by stable oligomer formation and by their weak fluorescence in vivo. Previous attempts to eliminate these problems by mutagenesis of RFP from Discosoma (DsRed) resulted in the monomeric mRFP1 and in the tetrameric RedStar RFP, which is distinguished by its enhanced fluorescence in vivo. Based on these mutations, we have generated an enhanced monomeric RFP, mRFPmars, and report its spectral properties. Together with green fluorescent labels, we used mRFPmars to visualize filamentous actin structures and microtubules in Dictyostelium cells. This enhanced RFP proved to be suitable to monitor the dynamics of cytoskeletal proteins in cell motility, mitosis, and endocytosis using dual-wavelength fluorescence microscopy.     

Selective fluorescent labeling of the 50-, 26-, and 20-kilodalton heavy chain segments of myosin ATPase

7-Diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin (CPI), rhodamine B isothiocyanate (RITC), and 4-bromomethyl-6,7-dimethoxycoumarin (BDMC), fluorescent reagents that can react covalently with amino or sulfhydryl groups, have been used to label myosin subfragment-1 (S-1) ATPase. The conditions under which CPI, RITC, and BDMC selectively label the 50-, 26-, and 20-kDa segments of the S-1 heavy chain, respectively, are described. CPI and RITC labeling little affects the ATPase activities of S-1 in the presence and absence of actin. BDMC labeling activates the Ca2+- and Mg2+-ATPases of S-1, and abolishes the K+-EDTA-ATPase. The three S-1 derivatives fluoresce strongly even under acidic conditions, suggesting the wide applicability of these fluorescent reagents as selective labels for the three segments of the S-1 heavy chain.     

Cyanine dye labeling reagents--carboxymethylindocyanine succinimidyl esters

Ten carboxymethylindocyanine dyes which form the basis of a new series of fluorescent probes have been synthesized and converted into succinimidyl active esters for fluorescent labeling of proteins or other amino-containing substances. Fluorescence emission maxima for members of the series range from 575 to 780 nm. Hydrophilic, water-soluble reagents have been obtained which yield labeled antibodies with little tendency to form precipitates. The fluorescence intensities achieved are higher than those produced by labeling with the cyanine isothiocyanates described previously (Mujumdar et al.: Cytometry 10:11-19, 1989). The utility of these reagents has been demonstrated in antibody labeling for two-color immunofluorescent imaging of internal structures in a mammalian cell and for two-color flow-cytometry experiments. The use of values of chromophore-equivalent weight (W/Ceq), calculated from quantitative absorption data on dye samples, is proposed as an aid in formulating labeling procedures.     

遗传密码扩充技术及其在蛋白质功能研究及标记成像中的应用

遗传密码扩充(genetic code expansion,GCE)技术利用终止密码子将非天然氨基酸掺入到蛋白质中,再结合点击反应对蛋白质实现定点标记.相较于荧光蛋白、标签抗体等其他标记工具,该技术在蛋白标记中使用的化合物分子较小、对蛋白空间结构影响较小,且能通过点击反应实现蛋白分子与染料分子1∶1的化学计量比,从而能...     

Tailor-made dyes for fluorescence correlation spectroscopy (FCS)

Two new fluorescent labels are presented that are optimized for excitation with He/Ne laser and red diode lasers. Application in FCS and labeling of proteins and oligomers are demonstrated. A strong rise of quantum yield and emission life time upon binding to biomolecules are characteristic features of the dyes.     

Accumulation of FlAsH/Lumio Green in active mitochondria can be reversed by β-mercaptoethanol for specific staining of tetracysteine-tagged proteins

Recent advances in the field of small molecule labels for live cell imaging promise to overcome some of the limitations set by the size of fluorescent proteins. We tested the tetracysteine–biarsenical labeling system in live cell fluorescence microscopy of reggie-1/flotillin-2 in HeLa and N2a cells. In both cell types, the biarsenical staining reagent FlAsH/Lumio Green accumulated in active mitochondria and led to mitochondrial swelling. This is indicative of toxic side effects caused by arsenic, which should be considered when this labeling system is to be used in live cell imaging. Mitochondrial accumulation of FlAsH/Lumio Green was reversed by addition of low concentrations of thiol-containing reagents during labeling and a subsequent high stringency thiol wash. Both ethanedithiol and β-mercaptoethanol proved to be effective. We therefore established a staining protocol using β-mercaptoethanol as thiol binding site competitor resulting in a specific staining of tetracysteine-tagged reggie-1/flotillin-2 of adequate signal to noise ratio, so that the more toxic and inconvenient ethanedithiol could be avoided. Furthermore, we show that staining efficiency was greatly enhanced by introducing a second tetracysteine sequence in tandem.M.F. Langhorst and S. Genisyuerek contributed equally to this work.     

The molecular properties and applications of Anthozoa fluorescent proteins and chromoproteins

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria and its fluorescent homologs from Anthozoa corals have become invaluable tools for in vivo imaging of cells and tissues. Despite spectral and chromophore diversity, about 100 cloned members of the GFP-like protein family possess common structural, biochemical and photophysical features. Anthozoa GFP-like proteins are available in colors and properties unlike those of A. victoria GFP variants and thus provide powerful new fluorophores for molecular labeling and intracellular detection. Although Anthozoa GFP-like proteins provide some advantages over GFP, they also have certain drawbacks, such as obligate oligomerization and slow or incomplete fluorescence maturation. In the past few years, effective approaches for eliminating some of these limitations have been described. In addition, several Anthozoa GFP-like proteins have been developed into novel imaging agents, such as monomeric red and dimeric far-red fluorescent proteins, fluorescent timers and photoconvertible fluorescent labels. Future studies on the structure of this diverse set of proteins will further enhance their use in animal tissues and as intracellular biosensors.     

Specific labeling of polypeptides at amino-terminal cysteine residues using Cy5-benzyl thioester

Even for moderately sized proteins, the multiple occurrence of cysteine and lysine residues often prevents the specific labeling of polypeptides with a single probe. To increase specificity, a method was developed to convert the commonly available succinimidyl esters of fluorescent dyes into benzyl thioesters via trimethyl aluminum-activated benzyl mercaptan. The thioester can then be reacted very specifically with polypeptides containing an N-terminal cysteine residue, forming a stable amide bond, analogous to the native chemical ligation of peptide fragments. Both reaction steps are easy to perform and proceed to high yields. The practicability of the approach was demonstrated using the popular cyanine dye Cy5 and a soluble peptide, and it is expected to be applicable to a wide range of succinimidyl esters and both chemically and recombinantly synthesized proteins. The method should dramatically facilitate the preparation of proteins for experiments requiring exact positioning of labels, for instance, F?rster resonance energy transfer studies.