GST Pull-Down Assay to Study PIF4 Binding In Vitro.

The phytochrome-interacting factor 4 (PIF4) is a well-known transcription factor that plays a pivotal role in plant thermomorphogenesis, coordinating growth and development in response to temperature changes. As PIF4 functions by forming complexes with other proteins, determining its interacting partners is essential for understanding its diverse roles in plant thermal responses. The GST (glutathione-S-transferase) pull-down assay is a widely used biochemical technique that enables the investigation of protein-protein interactions in vitro. It is particularly useful for studying transient or weak interactions between proteins. In this chapter, we describe the GST pull-down approach to detect the interaction between PIF4 and a known or suspected interacting protein. We provide detailed step-by-step descriptions of the assay procedures, from the preparation of recombinant GST-PIF4 fusion protein to the binding and elution of interacting partners. Additionally, we provide guidelines for data interpretation, quantification, and statistical analysis to ensure robust and reliable results.

Stain-Free Approach for Western Blot Analysis of Zebrafish Embryos.

Western blots are employed to detect and characterize amounts of proteins in biological samples. Quantifications are traditionally carried out through data normalization by housekeeping protein method. This approach does not account for variations not intrinsically dependent on the sample such as different experimental conditions and type of samples. Zebrafish researchers often face the challenge of comparing embryos at different developmental stages or from different strains. Housekeeping protein amount can change in these conditions therefore adding an unwanted quantification error. Here we describe the method to analyze mutant zebrafish embryos at different stages by western blot using the Stain-Free technology for normalization. We present Globin X quantification at 2 and 5 days postfertilization in wild type and in the bloodless Vlad Tepes (vlt) zebrafish mutant that lack red blood cells.

High-Throughput BN-PAGE for Mitochondrial Respiratory Complexes.

Blue native electrophoresis (BN-PAGE) is a highly resolutive method suited to the study of high molecular weight protein complexes between 100 and >3000 kDa. One of the drawbacks of this method is that it is very time-consuming and requires high quantities of purified organelles. Here we describe a high throughput BN-PAGE method allowing to screen libraries of plants potentially altered in respiratory metabolism.

Identification of lncRNA-Protein Interactions by CLIP and RNA Pull-Down Assays.

The emerging data indicates that long noncoding RNAs (lncRNAs) are involved in fundamental biological processes, and their deregulation may lead to oncogenesis and other diseases. LncRNA fulfil its biological functions at least in part by interacting with distinctive proteins. Here, we described two methods to identify the direct or indirect interactions between lncRNA and proteins: cross-linking and immunoprecipitation (CLIP) and RNA pull-down assay. CLIP methods enable yield a list of lncRNAs that directly interact target protein in living cells, whereas immunoprecipitation of biotin-labeled RNA (RNA pull-down) assay represents a method for identification of proteins that directly and indirectly bind with a particular target lncRNA of interest.

Validation of Isoform- and Mutation-Specific RAS Antibodies.

Validation of antibody specificity is essential for the accurate evaluation of protein expression. For antibodies that recognize the gene products of the RAS family of oncogenes (HRAS, KRAS, and NRAS), an important challenge is the determination of selectivity for the four nearly identical HRAS, KRAS4A, KRAS4B, and NRAS proteins. With increasing appreciation for the distinct roles of the different RAS proteins in normal and neoplastic cells, there is a need for well-validated antibodies to evaluate the function and expression of the different RAS isoforms. Here we describe our experimental approaches to characterize RAS antibodies for their isoform- and mutant-specificity for use in immunoblot analyses.

Detecting Necroptosis in Virus-Infected Cells.

Necroptosis has been implicated as a critical cell death pathway in cancers, Alzheimer's and other neurodegenerative diseases, and virus-infected cells. Necroptosis occurs when mixed-lineage kinase domain-like protein (MLKL) punctures the cytoplasmic membrane allowing a rapid influx of water leading to a loss of cellular integrity. As its role in human disease becomes apparent, methods identifying necroptosis will need to be further developed and optimized. Here we describe identification of necroptosis through quantifying cell death with pathway inhibitors and using western blots to identify end points of MLKL activation and protein-protein interactions leading to it.

Detection of Functionalized Sphingolipid Analogs in Detergent-Resistant Membranes of Immune Cells.

The analysis of protein enrichment in the detergent-resistant membranes (DRMs) isolated from immune cells enables us to analyze a link between the membrane lipid dynamics and cell activation. Here, we describe the fractionation of detergent-resistant membranes and the correlative analysis of the enrichment of T cell receptor (TCR) and ω-azido-modified synthetic ceramide in those fractions upon TCR stimulation.

Modified two-tiered testing algorithm for Lyme disease serology: the Canadian context.

BACKGROUND: Lyme disease (LD) is emerging in many parts of central and eastern Canada. Serological testing is most commonly used to support laboratory diagnosis of LD. Standard two-tiered testing (STTT) for LD involves detection of Borrelia burgdorferi antibodies using an enzyme immunoassay (EIA) followed by IgM and/or IgG immunoblots. However, improved sensitivity has been demonstrated using a modified two-tiered testing (MTTT) approach, in which a second EIA instead of the traditional immunoblot is used. This article summarises the evidence supporting the MTTT versus STTT for laboratory diagnosis of LD in Canada. METHODS: Peer reviewed literature on the sensitivity and specificity of different EIAs were compared by Canadian experts in LD diagnostic for MTTT vs STTT in patients with clinical history of LD residing in LD endemic areas or in samples from the LD serum repository. RESULTS: The MTTT approach consistently demonstrated improved sensitivity to detect early infections with B. burgdorferi and also maintained high specificity vs STTT. CONCLUSION: Diagnostic improvements in sensitivity of LD testing without significant loss of specificity have been consistently reported when MTTT is compared with STTT in studies conducted in highly LD endemic regions. Our working group agrees with the recommendation by the United States Centers for Disease Control that serological testing for LD using MTTT is an acceptable alternative to STTT. This recommendation is contingent on development and implementation of comprehensive validation studies on the performance of MTTT vs STTT within the Canadian context, including evaluation of the test performance in areas of low endemicity for LD.

Relación entre los anticuerpos contra antígenos extraíbles del núcleo y las enfermedades del tejido conectivo identificados por Immunoblots en un hospital y universidad de Lima / Relationship between antibodies against extractable nucleus antigens and connective tissue diseases identified by Immunoblot in a hospital in Lima, Peru

Objetivos: Determinar la relación de los anticuerpos con los antígenos del núcleo extraíble y las enfermedades del tejido conectivo identificadas por Immunoblot en un hospital de Lima, Perú. Material y métodos: Estudio de tipo observacional, ciencias básicas, analíticas y transversales, realizado en el Servicio de Inmunología del Hospital Nacional Arzobispo Loayza entre enero de 2018 y junio de 2018. Analizamos 291 historias clínicas de pacientes con enfermedad del tejido conectivo y para la detección de anticuerpos contra los antígenos extraíbles del núcleo se empleó el método de Immunoblots. Resultados: La frecuencia de los anticuerpos contra antígenos nucleares extraíbles en pacientes con enfermedad del tejido conectivo identificados por Immunoblot fue 789 (100%). Se demostró que existe una relación significativa p <0.05 de Anti-histonas (X2 = 64.19; p = 0,000), anti-nucleosomas (X2 = 71,16; p = 0,000), anti-dsDNA (X2 = 71,44; p = 0,000), anti-SM (X2 = 10,08; p = 0,003) y lupus eritematoso sistémico con prueba de Chi-cuadrado de Pearson. Se demostró que existe una relación significativa p <0.05 del Anti-SSA (X2 = 61,33; p = 0.001), anti-SSB (x2 = 51,00; p = 0.001), anti-Ro 52 (X2 = 62,60; p = 0,000) y síndrome de Sjogren con prueba de Chi-cuadrado de Pearson. Se demostró que existe una relación significativa p <0.05 de Anti-CENP B (p = 0.001) y calcinosis, fenómeno de Raynaud, dismotilidad esofágica, esclerodactilia y Telangiectasia (CREST) con Fisher. Conclusiones: Existe relación de anticuerpos con antígenos de núcleo extraíbles y lupus eritematoso sistémico, síndrome de Sjogren, enfermedad mixta del tejido conectivo, enfermedad del CREST, esclerodermia y polimiositis.

Objectives: To determine the relationship of antibodies to extractable nucleus antigens and connective tissue diseases identified by Immunoblot in a hospital in Lima, Peru. Material and methods: Study of the observational type, basic sciences, analytical and transversal, carried out in the Immunology service of the national Hospital Archbishop Loayza between January 2018 and June 2018. We analyzed 291 clinical histories of patients with connective tissue disease and for the detection of antibodies to the extractable antigens of the nucleus the method of Immunoblot was employed. Results: The frequency of the antibodies against extractable nuclear antigens in patients with connective tissue disease identified by Immunoblot was 789 (100%). It was demonstrated that there is significant relationship p < 0.05 of Anti-histones (X2 = 64.19; p = 0,000), anti-nucleosomas (X2 = 71,16; p = 0,000), anti-dsDNA (X2 = 71,44; p = 0,000), anti-SM (X2 = 10,08; p = 0,003) and Lupus Systemic erythematosus with Pearson Chi-square test. It was demonstrated that there is significant relationship p < 0.05 of the Anti-SSA (X2 = 61,33; p = 0.001), anti-SSB (X2 = 51,00; p = 0.001), anti-Ro 52 (X2 = 62,60; p = 0,000) and Sjogren's syndrome with Pearson Chi-square test. It was demonstrated that there is significant relationship p < 0.05 of Anti-CENP B (p = 0.001) and calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly and Telangiectasia (CREST) with exact Fisher statistician. Conclusions: There is a relationship of antibodies to extractable nucleus antigens and systemic lupus erythematosus, Sjogren's syndrome, mixed connective tissue disease, calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly and Telangiectasias (CREST), Scleroderma and Polymyositis.

Immunoblotting in Detection of Tumor-Associated Antigens in Esophageal Squamous Cell Carcinoma.

Tumor-associated antigens (TAAs) can be used as cancer markers and as signposts of therapeutic targets since their inimitable expression in cancer or significant overexpression in esophageal squamous cell carcinoma (ESCC) correlates with the initiation and progression of the diseases. Immunoblotting, also known as Western blotting or protein blotting, is a core technique in cell and molecular biology to detect proteins and glycoproteins. The technique allows detection of TAAs from complex protein samples such as in serum, aspirate, or solid tumor homogenate. In the process, proteins are separated according to the molecular weight. They were visualized within a gel matrix and then transferred to a supporting membrane. Finally, they are probed for binding with corresponding antibodies and identified the target proteins. Herein, we describe the Western blots analysis to detect protein or glycoprotein in samples from patients with esophageal squamous cell carcinoma (ESCC) or cells derived from ESCC.

Line Immunoblot Assay for Tick-Borne Relapsing Fever and Findings in Patient Sera from Australia, Ukraine and the USA.

Tick-borne relapsing fever (TBRF) is caused by spirochete bacteria of the genus Borrelia termed relapsing fever Borreliae (RFB). TBRF shares symptoms with Lyme disease (LD) caused by related Lyme disease Borreliae (LDB). TBRF and LD are transmitted by ticks and occur in overlapping localities worldwide. Serological detection of antibodies used for laboratory confirmation of LD is not established for TBRF. A line immunoblot assay using recombinant proteins from different RFB species, termed TBRF IB, was developed and its diagnostic utility investigated. The TBRF IBs were able to differentiate between antibodies to RFB and LDB and had estimated sensitivity, specificity, and positive and negative predictive values of 70.5%, 99.5%, 97.3%, and 93.4%, respectively, based on results with reference sera from patients known to be positive and negative for TBRF. The use of TBRF IBs and analogous immunoblots for LD to test sera of patients from Australia, Ukraine, and the USA with LD symptoms revealed infection with TBRF alone, LD alone, and both TBRF and LD. Diagnosis by clinical criteria alone can, therefore, underestimate the incidence of TBRF. TBRF IBs will be useful for laboratory confirmation of TBRF and understanding its epidemiology worldwide.

Detection of MLKL Oligomerization During Programmed Necrosis.

Programmed necrosis, also known as necroptosis, is a form of regulated necrotic cell death that is mediated by receptor-interacting protein kinases RIP1 (or RIPK1), RIP3 (or RIPK3), and the mixed lineage kinase domain-like protein, MLKL. Following the induction of programmed necrosis, MLKL is phosphorylated by RIP3 and oligomerizes and then the protein translocates to cell plasma membrane in order to execute programmed necrosis. Here, we describe a detailed protocol to detect MLKL oligomerization in necroptotic cells by Western blotting analysis under nonreducing condition. Therefore, we established the method to detect the activation of programmed necrotic pathway.

Western Blot Methodologies for Analysis of In Vitro Protein Expression Induced by Teratogenic Agents.

Western blotting permits immunodetection, characterization, and quantification of proteins in cell (or tissue) homogenates. It also enables detection of protein modification (e.g., phosphorylation) or degradation (e.g., hydrolysis), even at low abundance. Sodium dodecyl sulfate (SDS)-polyacrylamide gel is used to separate proteins from homogenate which are then transferred electrophoretically to polyvinylidene difluoride (PVDF) membranes. After membrane "blocking," to reduce nonspecific binding, proteins of interest are detected using specific antibodies (antigen detection), which are then bound to a secondary antibody linked to a label (e.g., fluorescent, chemiluminescent, or chromophore). After signal detection and acquisition, quantification of the resulting bands is achieved using densitometry software. Results are normalized against controls and housekeeping proteins (e.g., GAPDH, beta-actin and tubulin), which are constitutively expressed proteins that maintain cell viability. This chapter outlines the use of the Western blot technique optimized for the in vitro analysis of changes in the protein expression induced by teratogenic exposure.

Combining 2-DE immunoblots and mass spectrometry to identify putative soybean (Glycine max) allergens.

Soybean is recognized as a commonly allergenic food, but the identity of important allergens is not well studied. Recently, some global regulatory agencies started requiring quantitative analysis of individual allergens, including unproven allergens, as part of the risk assessment for genetically engineered (GE) soybeans. We sought to identify soybean proteins that bind IgE from any of 10 individual soybean-sensitized subjects. Soybean IgE binding proteins were identified by 2-DE immunoblots using sera from four soy-allergic and plasma from six soy-sensitized human subjects. Corresponding spots were excised from stained gels, digested, and analyzed using a quadrupole TOF Synapt G2-S tandem mass spectrometer. Results showed the major IgE binding proteins were subunits of either ß-conglycinin (Gly m 5) or glycinin (Gly m 6). Soybean Kunitz trypsin inhibitor (SKTI) was a significant IgE binding protein for four subjects. Soybean agglutinin, seed biotinylated protein (SBP) of 65 kDa, late embryogenesis protein (LEP), and sucrose-binding protein were identified as IgE binding only for soy-sensitized subjects. We conclude that the major soybean allergens are isoforms of Gly m 5, Gly m 6, and possibly SKTI and that requirements for quantitative measurement of proteins that are not clear allergens is not relevant to safety.

Autophagic Flux Assessment in Colorectal Cancer Cells.

Autophagy protects colorectal cancer cells against therapeutic intervention. Autophagy is a continuous process, and autophagic flux requires both autophagosome synthesis and their subsequent degradation at lysosomes. Hence, cells with elevated autophagic flux display both rapid autophagosome generation and degradation. Here, we describe an immunoblot protocol coupled to pharmaceutical inhibition of autophagosome clearance to monitor autophagic flux levels between colorectal cancer cell lines.

Study of Autophagy in Plant Senescence.

As a major intracellular degradation pathway, autophagy contributes to nutrient recycling and is indispensable during plant senescence. Here we describe methods used for investigating the autophagic process during leaf senescence. These include transcript analysis of core machinery autophagy genes, immunoblotting of ATG8, and microscopic observation of autophagosome formation.

Electrophoresis- and FRET-Based Measures of Serpin Polymerization.

Many serpinopathies, including alpha-1 antitrypsin (A1AT) deficiency, are associated with the formation of unbranched polymer chains of mutant serpins. In vivo, this deficiency is the result of mutations that cause kinetic or thermodynamic destabilization of the molecule. However, polymerization can also be induced in vitro from mutant or wild-type serpins under destabilizing conditions. The characteristics of the resulting polymers are dependent upon induction conditions. Due to their relationship to disease, serpin polymers, mainly those formed from A1AT, have been widely studied. Here, we describe Förster resonance energy transfer (FRET) and gel-based approaches for their characterization.

Immunoprecipitation of Death Inducing Signaling Complex by Caspase-8.

Analysis of CD95/Fas complexes by immunoprecipitation has long relied on the monoclonal antibody APO1 or tagged recombinant Fas ligand. Immunoprecipitation is an elegant and efficient procedure to investigate endogenous protein interactions or complexes. Provided that the targeted complex is soluble in mild detergent these complexes can be recovered using protein A/G-coupled Sepharose beads and further analyzed after denaturation and electrophoretic separation by western blotting or mass spectrometry. Herein, we describe in detail the method used in our laboratory to immunoprecipitate and analyze by immunoblot complexes containing caspase-8, using a commercial antibody directed against caspase-8.

Microwestern Arrays for Systems-Level Analysis of SH2 Domain-Containing Proteins.

The Microwestern Array (MWA) method combines the scalability and miniaturization afforded by the Reverse Phase Lysate Array (RPLA) approach with the electrophoretic separation characteristic of the Western blot. This technology emulates the creation of an array of small Western blots on a single sheet of nitrocellulose allowing for the sensitive and quantitative measurement of hundreds of proteins from hundreds of cell lysates with minimal cost and maximal accuracy, precision, and reproducibility. The MWA is a versatile technology that can be easily configured for purposes such as antibody screening, cell signaling network inference, protein modification/phenotype regression analysis, and genomic/proteomic relationships. Accordingly, configurations for the MWA can be optimized for maximal numbers of proteins analyzed from small numbers of cell lysates, for small numbers of antibodies against large numbers of cell lysates, or for maximal resolution of protein size achieved by increased electrophoretic separation distance. For example, on a single gel, 6 samples can be printed 96 times if a few samples need to be assayed with a large number of antibodies. Alternatively, up to 100 samples can be assayed with four antibodies on a single gel. Intermediate configurations are also discussed.The efficiency of the MWA is orders of magnitude greater in reagents, labor, and time required per data point relative to the standard Western blotting method and orders of magnitude more sensitive than standard mass spectrometry methods. The MWA is therefore a very attractive approach for capturing global changes in protein abundances and modifications including tyrosine phosphorylation and SH2 domain binding sites.

Proteomic Analysis Reveals the Dynamic Role of Silicon in Alleviation of Hyperhydricity in Carnation Grown In Vitro.

The present study depicted the role of silicon in limiting the hyperhydricity in shoot cultures of carnation through proteomic analysis. Four-week-old healthy shoot cultures of carnation "Purple Beauty" were sub-cultured on Murashige and Skoog medium followed with four treatments, viz. control (-Si/-Hyperhydricity), hyperhydric with no silicon treatment (-Si/+Hyperhydricity), hyperhydric with silicon treatment (+Si/+Hyperhydricity), and only silicon treated with no hyperhydricity (+Si/-Hyperhydricity). Comparing to control morphological features of hyperhydric carnations showed significantly fragile, bushy and lustrous leaf nature, while Si supply restored these effects. Proteomic investigation revealed that approximately seventy protein spots were differentially expressed under Si and/or hyperhydric treatments and were either up- or downregulated in abundance depending on their functions. Most of the identified protein spots were related to stress responses, photosynthesis, and signal transduction. Proteomic results were further confirmed through immunoblots by selecting specific proteins such as superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), PsaA, and PsbA. Moreover, protein-protein interaction was also performed on differentially expressed protein spots using specific bioinformatic tools. In addition, stress markers were analyzed by histochemical localization of hydrogen peroxide (H2O2) and singlet oxygen (O21-). In addition, the ultrastructure of chloroplasts in hyperhydric leaves significantly resulted in inefficiency of thylakoid lamella with the loss of grana but were recovered in silicon supplemented leaves. The proteomic study together with physiological analysis indicated that Si has a substantial role in upholding the hyperhydricity in in vitro grown carnation shoot cultures.