Labeling Procedure Research Articles

Tyramide signal amplification as universal detection method on protein coated microbeads

Quantitative analysis of a target molecule in a microbead-based fluorescent assay requires a specific labeling procedure. For nucleic acid analysis the hybridization with florescent labeled oligonucleotides is the most common method. However, disadvantages are the necessity for direct labeling of p robes and the sensitivity to detect low amounts of target molecules. In this study we established an alternative detection method for biomolecules on microbeads, the tyramide signal amplification (TSA). Hereby, biomolecules are detected by enzymatically activated and fluorophore-conjugated tyramides that bind to specific protein residues. This method has proven to be a versatile and robust enzyme amplification technique for sensitive immunohistochemical detection. Now, we present the feasibility of the TSA procedure to detect hybridized biotinylated oligonucleotide probes bound to protein coated microbead surfaces TSA was performed using fluorescent, size-encoded and streptavidin coated microbeads that were loaded with dual-biotinylated DNA capture probes, prepared from polymerase chain reaction. Beside streptavidin alone for surface coating of those microbeads, we applied different quantities of streptavidin in combination with bovine serum albumin, immunoglobulin G or Protein G/A, to check for positive effects on the resulting signal intensities through specific binding of tyramide molecules. For this method, streptavidin turned out as appropriate protein for the surface binding, without the need for further molecules. In comparison to a standard detection with common streptavidin-fluorophore-conjugates TSA showed its advantage in the detection of low probe amounts down to a concentration of 3.3·10-4 ng/μL.

Fluorescent labelling of boar spermatozoa for quantitative studies on competitive sperm-oviduct binding.

Invitro sperm-oviduct binding assays enable assessment of the capacity of spermatozoa to form a 'reservoir' in the oviduct. Competitive approaches, such as experimental set-ups that test multiple males or semen samples simultaneously on the same tissue explants, are desirable because they reduce the likelihood of bias when using material from different females. Therefore, we established a fluorescent labelling technique that allows tagging and storage of spermatozoa before competitive studies of sperm-oviduct binding invitro. Fluorescent markers were tested for reliability and compatibility with parameters of boar spermatozoa viability. The addition of seminal plasma after density gradient centrifugation was essential to counteract centrifugation stress during the labelling procedure. It was demonstrated that sperm tagged with MitoTracker Green FM or MitoTracker Red FM can be successfully used in competitive sperm-oviduct binding studies. The assay was sensitive enough to indicate subtle effects of semen storage temperature on the ability of the spermatozoa to contribute to the female sperm reservoir.

DEER distance measurements on trityl/trityl and Gd(iii)/trityl labelled proteins.

Triarylmethyl (TAM or trityl) radicals are becoming important for measuring distances in proteins and nucleic acids. Here, we report on a new trityl spin label CT02MA, which conjugates to a protein via a redox stable thioether bond. The performance of the new spin label was demonstrated in W-band double electron-electron resonance (DEER) distance measurements on doubly trityl-labelled mutants of immunoglobulin G-binding protein 1 (GB1) and ubiquitin. For both doubly CT02MA-labelled proteins we measured, by applying chirped pump pulse(s), relatively narrow distance distributions, comparable to those obtained with the same protein mutants doubly labelled with BrPy-DO3MA-Gd(iii). We noticed, however, that the sample contained some free CT02MA that was difficult to remove at the purification step. Dual labelling of ubiquitin with one CT02MA tag and one BrPy-DO3MA-Gd(iii) tag was achieved as well and the trityl-Gd(iii) distance distribution was measured, facilitated by the use of a dual mode cavity in combination with a chirped pump pulse. We also measured the Gd(iii)-Gd(iii) distance distribution in this sample, showing that the labelling procedure was not fully selective. Nevertheless, these measurements demonstrate the potential of the high sensitivity Gd(iii)-trityl W-band DEER distance measurements in proteins, which can be further exploited by designing orthogonal Gd(iii)/trityl labelling schemes.

Unlabeled imaging of primary cilia by scanning ion conductance microscopy

Primary cilia are non-motile cilia consisting of a centriole-derived basal body and a microtubule-based axoneme. In recent years, the structure and function of primary cilia have been attracting attention due to the relation with the onset of ciliary disease. Scanning ion conductance microscopy (SICM) is a probe microscopy used to measure the topography and functions of living cells at nanoscale. Furthermore, the labelling procedure is not necessary for SICM measurement compare to fluorescence imaging. We compared the structures of primary cilia of human retinal pigment epithelial cell line (RPE-1 cells) and Madin-Darby canine kidney cell line (MDCK cells) at nanoscale by using SICM. In addition, high resolution SICM images have also succeeded in visualizing ciliary pockets that difficult to be fluorescently labeled.

Site-Specific Antibody Labeling Using Phosphopantetheinyl Transferase-Catalyzed Ligation.

4'-Phosphopantetheinyl transferases (PPTases) have been employed by researchers as versatile biocatalysts for the site-specific modification of numerous protein targets with structurally diverse molecules. Here we describe the use of these enzymes for the production of homogeneous antibody-drug conjugates (ADCs), which have garnered much attention as innovative anticancer drugs. The exceptionally broad substrate tolerance of PPTases allows for one-step and two-step conjugation strategies for site-specific ADC synthesis. While one-step conjugation involves direct coupling of a drug molecule to an antibody, two-step conjugation provides increased flexibility and efficiency of the conjugation process by first attaching a bioorthogonal chemical handle that is then used for drug molecule attachment in a second step. The aim of this chapter is to outline detailed protocols for both labeling procedures, as well as to provide guidance on enzyme and substrate preparation.

Photoaffinity Labeling of Respiratory Complex I in Bovine Heart Submitochondrial Particles by Photoreactive [125I] amilorides.

The architecture of quinone/inhibitor-access channel in proton-translocating NADH-quinone oxidoreductase (respiratory complex I) was modeled by X-ray crystallography and cryo-EM, however, it remains debatable whether the channel model reflects the physiologically relevant state present throughout the catalytic cycle. Using photoreactive [125I]amilorides, we demonstrated that amiloride-type inhibitors bind to the interfacial region of multiple subunits (49-kDa, ND1, PSST, and 39-kDa subunits), which is difficult to reconcile with the current channel model. This report describes the procedures for photoaffinity labeling of bovine submitochondrial particles by photoreactive [125I]amilorides. The protocol could be widely applicable for the characterization of various biologically active compounds, whose target protein remains to be identified or characterized.

Metabolic Labeling of Cultured Mammalian Cells for Stable Isotope-Resolved Metabolomics: Practical Aspects of Tissue Culture and Sample Extraction.

Stable isotope-resolved metabolomics (SIRM) methods are used increasingly by cancer researchers to probe metabolic pathways and identify vulnerabilities in cancer cells. Analytical and computational advances are being made constantly, but tissue culture and sample extraction procedures are often variable and not elaborated in the literature. This chapter discusses basic aspects of tissue culture practices as they relate to the use of stable isotope tracers and provides a detailed metabolic labeling and metabolite extraction procedure designed to maximize the amount of information that can be obtained from a single tracer experiment.

Immunophenotyping of Acute Lymphoblastic Leukemia.

Immunophenotyping by flow cytometry is an important component in the diagnostic evaluation of patients with acute lymphoblastic leukemia. This technique further permits the detection of minimal residual disease after therapy, a robust prognostic factor that may guide individualized treatment. We describe here laboratory methods for both the initial characterization of lymphoblasts at diagnosis, and the detection of rare leukemic lymphoblasts after treatment. In addition to antibody combinations suitable for diagnosis and detection of minimal residual disease, we describe procedures for peripheral blood and bone marrow sample preparation, procedures for labeling of cell-surface and intracellular proteins with fluorochrome-conjugated antibodies, and approaches to analysis of immunophenotypic data.

Site-Selective Enzymatic Labeling of Designed Ankyrin Repeat Proteins Using Protein Farnesyltransferase.

Affinity agents coupled to a functional moiety play an ever-increasing role in modern medicine, ranging from radiolabeled selective binders in diagnosis to antibody-drug conjugates in targeted therapies. In biomedical research, protein coupling to fluorophores, surfaces and nanoparticles has become an integral part of many procedures. In addition to antibodies, small scaffold proteins with similar target binding properties are being widely explored as alternative targeting moieties. To label these binders of interest with different functional moieties, conventional chemical coupling methods can be employed, but often result in heterogeneously modified protein products. In contrast, enzymatic labeling methods are highly site-specific and efficient. Protein farnesyltransferase (PFTase) catalyzes the transfer of an isoprenoid moiety from farnesyl diphosphate (FPP) to a cysteine residue in a C-terminal CaaX motif at the C-terminus of a protein substrate. The addition of only four amino acid residues minimizes the influence on the native protein structure. In addition, a variety of isoprenoid analogs containing different bioorthogonal functional groups, including azides, alkynes, and aldehydes, have been developed to enable conjugation to various cargos after being incorporated onto the target protein by PFTase. In this protocol, we present a detailed procedure for labeling Designed Ankyrin Repeat Proteins (DARPins) engineered with a C-terminal CVIA sequence using an azide-containing FPP analog by yeast PFTase (yPFTase). In addition, procedures to subsequently conjugate the labeled DARPins to a TAMRA fluorophore using strained-promoted alkyne-azide cycloaddition (SPAAC) reactions as well as the sample preparation to evaluate the target binding ability of the conjugates by flow cytometry are described.

Probing Alpha-Synuclein Conformations by Electron Paramagnetic Resonance (EPR) Spectroscopy.

Electron paramagnetic resonance (EPR) spectroscopy in combination with site-directed spin labeling is ideally suited to study structure, dynamics, and interactions of intrinsically disordered proteins as alpha-synuclein.Here we describe all steps required for a corresponding study: the spin labeling procedure, sample preparation, spectroscopic experimental procedure, and data analysis.

Cell surface pH imaging using poly(ethylene glycol)-phospholipid: its potential as the core structure of membrane anchored-probes

Various physiological and pathological processes are accompanied with the local acidification of extracellular local pH. However, imaging tools to investigate the spatio-temporal dynamics as well as the functional significance of cell surface pH are limitedly available. We established a novel method of in vitro cell surface pH imaging by using a membrane-anchored pH probe, poly(ethylene glycol)-phospholipid conjugated with fluorescein isothiocyanate (FITC-PEG-lipid). PEG-lipid, amphiphilic synthetic polymer, is a biomaterial originally synthesized for cell-surface engineering for transplantation therapy. When added into the cell culture medium, FITC-PEG-lipid was spontaneously inserted into the plasma membrane via its phospholipid moiety. FITC-PEG-lipid was retained at the extracellular surface due to the hydrophobic PEG moiety. The ratiometric readout of FITC fluorescence was unique to the extracellular pH in the range of weakly alkaline and acidic pH (pH 5.0-7.5). The pH measurement with FITC-PEG-lipid was accurate enough to distinguish the difference of 0.1 pH unit for the external solutions at pH 5.9, 6.0 and 6.1, near the inflection point of fluorescence ratio. The response of FITC-PEG-lipid to the extracellular pH was reversible. Continuous alteration of extracellular pH was successfully visualized by time-lapse imaging analysis. Our study demonstrated that FITC-PEG-lipid is useful as a sensitive and reversible cell surface-anchored pH probe. The simple labeling procedure of FITC-PEG-lipid is advantageous especially when considering its application to high-throughput in vitro assay. Furthermore, PEG-lipid holds a great potential as the membrane anchor of various analytical probes to approach the juxtamembrane environments.

A Distinctive MRI-Based Absolute Bias Correction Protocol for the Potential Labelling and In Vivo Tracking of Stem Cells in a TBI Mice Model.

Traumatic brain injury (TBI) is a leading cause of death and disability. The condition is difficult to treat owing to its heterogeneous nature and complex biological pathways. Stem cell transplantation is an emerging self-deliverable therapeutic modality which could immensely improve the invigorating management of the problem. The synergistic interaction of the stem cells with the paracrine niche molecules at the site of injury is an end point that decides the cells' effective tissue-forming regenerative response. Thus, noninvasive monitoring and tracking of the infused stem cells is quite decisive after transplantation. Here, we have designed and validated a distinctive in vivo magnetic resonance imaging protocol to monitor the transplanted mesenchymal stem cells (MSCs) longitudinally in TBI-induced mice. We have further described the synthesis of improved transverse relaxivity contrast agent, a protocol for the efficient labelling of MSCs, preparation of a TBI model system in mice, and the imaging and tracking of the implanted stem cells at the injury site through 7T MRI. MGE-T2∗ imaging in association with relaxometry-based quantitative assessment using absolute bias correction provided a suitable mechanism to monitor and track the infused labelled stem cells at the TBI site. High transverse relaxivity negative contrast agent synthesis, MSC labelling procedure, and quantitative T2∗ time measurement normalized with absolute bias correction are the key features of this protocol. This procedure has immense application potential and could therefore be extrapolated to stem cell tracking during the treatment of various diseases.

Validation of in vitro labeling method for human use of heat-damage red blood cells to detect splenic tissue and hemocateretic function.

Background and aim: Selective imaging of the splenic tissue is obtained with heat-damaged, or heat-denatured, red blood cells (RBCs) of the patient labeled with 99mTc in a variety of clinical scenarios. Aim of the study was to validate the process used for labelling heat-damaged red blood cells “totally in vitro”, after blood sample collection, before re-inject labeled RBCs to the patient. Moreover, we assessed efficacy of the staff training programme in order to guarantee repeatibility and method standardization in the clinical routine. Methods: The validation process of the labeling procedure was performed in three different patients during three consecutive days. After collection of a blood sample using a heparinized syringe, we isolated erythrocytes from other blood components by centrifugation and washing steps. Then, we added the stannous pyrophosphate (PYP) to the erythrocytes pellet, after pH control. The ‘pretinning’ of RBCs was necessary to reduce Tc-99m once pertechnetate was entered them. After the labeling reaction with 130 MBq of 99mTc-pertechnetate, the erythrocytes were denatured in a water bath at a temperature of 49°-50°C, for 10 min. Radioactivity of blood aliquotes was measured with a dose calibrator and labelling efficiency (LE%) was determined. The labelling purity was measured using a gamma counter and calculated using the formula: counts of pellet/counts of pellet+(counts of surnatant)*100. Training program was evaluated using a Learning Questionnaire (LQ). with a grading score from 6 (“”) to 1 (“nothing”) for each operator (n=3). Results: We didn’t observed the presence of macroaggregates during the entire process, until the final sample. The labelling efficiency resulted at very high values in the three consecutive measured aliquotes (mean value 73.67%) as well as the labelling purity (>95.22%). In our instituion, we use splenic imaging with labelled heat-damaged RBCs to detect ectopic spleen, splenosis, extramedullary hematopoiesis. We performed 3 procedures with heat-damaged labeled RBCs with a mean labelling efficiency 73.67%. Training and learning programmes were scored by key objective areas with a mean value of 5. Conclusions: Our in vitro labeling process of heat-damaged RBCs is simple and safe, providing a useful technique easy to implement in clinical routine for splenic imaging Learning outcome of the training programme was scored as effective by all the operators with evidence of high-efficiency-reproducible procedure mantained over time. (www.actabiomedica.it)

Discrimination of normal skin fibroblast and malignant melanocytes using dielectrophoretic force and fluid-induced shear force

Cell analysis is an important technology that is widely used for medical diagnosis in hospitals and cell engineering research. Among cell analysis technology, dielectrophoresis (DEP) is one of the most promising approaches for discriminating biological particles because this phenomena requires no labeling procedure with a fluorescent dye or magnetic beads. In this study, we developed a precise cell analysis system by evaluating the DEP force with a liquid flow system. The DEP forces acting on a cell was characterized using a microfluidic chamber containing an electrode-array and fluid-induced shear forces. On the basis of this characterization, the discrimination between healthy skin cells and cancer cells was performed using our novel DEP cell sorting system. As a result, the healthy cells and cancer cells could be distinguished even if the dielectrophoretic properties of those cells were similar.

High-resolution Immunoelectron Microscopy Techniques for Revealing Distinct Subcellular Type 1 Cannabinoid Receptor Domains in Brain.

Activation of type 1 cannabinoid (CB1) receptors by endogenous, exogenous (cannabis derivatives) or synthetic cannabinoids (i.e., CP 55.940, Win-2) has a wide variety of behavioral effects due to the presence of CB1 receptors in the brain. In situ hybridization and immunohistochemical techniques have been crucial for defining the CB1 receptor expression and localization at the cellular level. Nevertheless, more advanced methods are needed to reveal the precise topography of CB1 receptors in the brain, especially in unsuspected sites such as other cell types and organelles with low receptor expression (e.g., glutamatergic neurons, astrocytes, mitochondria). High-resolution immunoelectron microscopy provides a more precise detection method for the subcellular localization of CB1 receptors in the brain. Herein, we describe a single pre-embedding immunogold method for electron microscopy based on the use of specific CB1 receptor antibodies and silver-intensified 1.4 nm gold-labeled Fab' fragments, and a combined pre-embedding immunogold and immunoperoxidase method that employs biotinylated secondary antibodies and avidin-biotin-peroxidase complex for the simultaneous localization of CB1 receptors and protein markers of specific brain cells or synapses (e.g., GFAP, GLAST, IBA-1, PSD-95, gephyrin). In addition, a post-embedding immunogold method is also described and compared to the pre-embedding labeling procedure. These methods provide a relatively easy and useful approach for revealing the subcellular localization of low amounts of CB1 receptors in glutamatergic synapses, astrocytes, neuronal and astrocytic mitochondria in the brain.

Information extraction using neural language models for the case of online job listings analysis

In this article we discuss the approach to information extraction (IE) using neural language models. We provide a detailed overview of modern IE methods: both supervised and unsupervised. The proposed method allows to achieve a high quality solution to the problem of analyzing the relevant labor market requirements without the need for a time-consuming labelling procedure. In this experiment, professional standards act as a knowledge base of the labor domain. Comparing the descriptions of work actions and requirements from professional standards with the elements of job listings, we extract four entity types. The approach is based on the classification of vector representations of texts, generated using various neural language models: averaged word2vec, SIF-weighted averaged word2vec, TF-IDF-weighted averaged word2vec, paragraph2vec. Experimentally, the best quality was shown by the averaged word2vec (CBOW) model.

An efficient method for FITC labelling of proteins using tandem affinity purification.

Fluorescence-based assays are extremely diverse, sensitive and robust experimental methods for investigating the conformational changes, enzyme kinetics, dynamics and molecular interactions. A prerequisite for most of these experimental approaches is to label the protein of interest with one or more extrinsic fluorophores with desired photophysical properties. Fluorescein isothiocyanate (FITC), due to its high quantum efficiency and conjugate stability, is most widely used fluorescence labelling reagent for such experimental approaches. However, the bottlenecks in this labelling reaction is requirement of high protein concentration, maintenance of protein stability during the labelling process as well as high background fluorescence due to ineffective removal of unreacted FITC, prior to fluorescence studies. Therefore, to overcome these inadequacies or limitations, we have modified the existing protocol by introducing tandem affinity purification tags at the N- and C-terminus of target protein. Using this modified method, we have efficiently labelled target protein with significant decrease in precipitation, degradation and background fluorescence of unreacted FITC. This facile and rapid technique may also be used as a basis for labelling procedures with other fluorophores and hence has a broad application in spectroscopic studies.

Fully Automated 89Zr Labeling and Purification of Antibodies.

Dozens of monoclonal antibodies (mAbs) have been approved for clinical use, and hundreds more are under development. To support these developments and facilitate a personalized medicine approach, PET imaging and quantification of mAbs, after chelation with desferrioxamine B (DFO) and radiolabeling with 89Zr, has become attractive. Also, the use of 89Zr-mAbs in preclinical and clinical studies is expanding rapidly. Despite these rapid developments, 89Zr radiolabeling is still performed manually. Therefore, we aimed to develop a simple, fully automated, good-manufacturing-practice (GMP)-compliant production procedure for the 89Zr labeling of mAbs. Such procedures should increase the robustness and capacity of 89Zr-mAb production while minimizing the radiation dose to the operator. Here, the procedures for fully automated 89Zr-mAb production are described and applied to produce batches of 89Zr-DFO-N-suc-cetuximab and 89Zr-DFO-N-suc-rituximab suitable for clinical use. Both products had to meet the GMP-compliant quality standards with respect to yield, radiochemical purity, protein integrity, antigen binding, sterility, and endotoxin levels. Methods: Automated 89Zr labeling of mAbs was developed on a Scintomics GRP 2V module and comprised the following steps: reagent transfer to the 89Zr-containing reaction vial, mixing of the reagents followed by a 60-min reaction at room temperature to obtain optimal radiolabeling yields, and product purification using a PD-10 desalting column. Results: Radiochemical yields of 89Zr-DFO-N-suc-cetuximab and 89Zr-DFO-N-suc-rituximab were all more than 90% according to instant thin-layer chromatography. Isolated yields were 74.6% ± 2.0% and 62.6% ± 3.0% for 89Zr-DFO-N-suc-cetuximab and 89Zr-DFO-N-suc-rituximab, respectively, which are similar to isolated yields obtained using GMP protocols for manual 89Zr labeling of mAbs. To meet the GMP-compliant quality standards, only the radiochemically pure fractions were collected from PD-10, resulting in a lower isolated yield than the radiochemical yield according to instant thin-layer chromatography. The radiochemical purity and protein integrity were more than 95% for both products, and the antigen binding was 95.6% ± 0.6% and 87.1% ± 2.2% for 89Zr-DFO-N-suc-cetuximab and 89Zr-DFO-N-suc-rituximab, respectively. The products were sterile, and the endotoxin levels were within acceptable limits, allowing future clinical production using this procedure. Conclusion: Procedures for fully automated GMP-compliant production of 89Zr-mAbs were developed on a commercially available synthesis module, which also allows the GMP production of other radiolabeled mAbs.

Spatial Referencing of Hyperspectral Images for Tracing of Plant Disease Symptoms

The characterization of plant disease symptoms by hyperspectral imaging is often limited by the missing ability to investigate early, still invisible states. Automatically tracing the symptom position on the leaf back in time could be a promising approach to overcome this limitation. Therefore we present a method to spatially reference time series of close range hyperspectral images. Based on reference points, a robust method is presented to derive a suitable transformation model for each observation within a time series experiment. A non-linear 2D polynomial transformation model has been selected to cope with the specific structure and growth processes of wheat leaves. The potential of the method is outlined by an improved labeling procedure for very early symptoms and by extracting spectral characteristics of single symptoms represented by Vegetation Indices over time. The characteristics are extracted for brown rust and septoria tritici blotch on wheat, based on time series observations using a VISNIR (400–1000 nm) hyperspectral camera.

Evaluation and optimisation of unnatural amino acid incorporation and bioorthogonal bioconjugation for site-specific fluorescent labelling of proteins expressed in mammalian cells

Many biophysical techniques that are available to study the structure, function and dynamics of cellular constituents require modification of the target molecules. Site-specific labelling of a protein is of particular interest for fluorescence-based single-molecule measurements including single-molecule FRET or super-resolution microscopy. The labelling procedure should be highly specific but minimally invasive to preserve sensitive biomolecules. The modern molecular engineering toolkit provides elegant solutions to achieve the site-specific modification of a protein of interest often necessitating the incorporation of an unnatural amino acid to introduce a unique reactive moiety. The Amber suppression strategy allows the site-specific incorporation of unnatural amino acids into a protein of interest. Recently, this approach has been transferred to the mammalian expression system. Here, we demonstrate how the combination of unnatural amino acid incorporation paired with current bioorthogonal labelling strategies allow the site-specific engineering of fluorescent dyes into proteins produced in the cellular environment of a human cell. We describe in detail which parameters are important to ensure efficient incorporation of unnatural amino acids into a target protein in human expression systems. We furthermore outline purification and bioorthogonal labelling strategies that allow fast protein preparation and labelling of the modified protein. This way, the complete eukaryotic proteome becomes available for single-molecule fluorescence assays.