Blotting Membrane Research Articles

RNA Blotting by Electrotransfer and RNA Detection.

Northern blot, or RNA blot, is a widely used technique in molecular biology to detect and semi-quantify specific RNAs. The advantage of this method is its ability to simultaneously estimate the sizes and quantities of degraded or processed RNA products. Northern blotting involves the use of electrophoresis to separate RNA samples by size. These RNAs are then transferred and immobilized on a membrane, and the RNAs of interest are detected by hybridization using a sequence-specific labeled DNA probe. Agarose gels are typically used in routine procedures to allow for the separation and specific detection of high molecular weights (ranging from tRNA size to several kb RNAs). However, acrylamide gels are preferred when the separation of lower weight RNAs (ranging from 20 to 200 nucleotides) is required. Both approaches for RNA separation are presented here. The term "northern blot" originally referred to the capillary transfer of RNA from the electrophoresis gel to the blotting membrane. However, RNA transfer to the membrane by electrotransfer has also been proven to be a suitable and fast method for transferring RNAs from acrylamide and agarose gels to the membrane.

Synergistic effects of quercetin-loaded CoFe2O4@Liposomes regulate DNA damage and apoptosis in MCF-7 cancer cells: based on biophysical magnetic hyperthermia

Introduction Breast cancer (BC) is the most common malignancy in women globally. Significant progress has been made in developing structural nanoparticles (NPs) and formulations for targeted smart drug delivery (SDD) of pharmaceuticals, improving the precision of tumor cell targeting in therapy. Significance Magnetic hyperthermia (MHT) treatment using magneto-liposomes (MLs) has emerged as a promising adjuvant cancer therapy. Methods CoFe2O4 magnetic NPs (MNPs) were conjugated with nanoliposomes to form MLs, and the anticancer drug quercetin (Que) was loaded into MLs, forming Que-MLs composites for antitumor approach. The aim was to prepare Que-MLs for DD systems (DDS) under an alternating magnetic field (AMF), termed chemotherapy/hyperthermia (chemo-HT) techniques. The encapsulation efficiency (EE), drug loading capacity (DL), and drug release (DR) of Que and Que-MLs were evaluated. Results The results confirmed successful Que-loading on the surface of MLs, with an average diameter of 38 nm and efficient encapsulation into MLs (69%). In vitro, experimental results on MCF-7 breast cells using MHT showed high cytotoxic effects of novel Que-MLs on MCF-7 cells. Various analyses, including cytotoxicity, apoptosis, cell migration, western blotting, fluorescence imaging, and cell membrane internalization, were conducted. The Acridine Orange-ethidium bromide double fluorescence test identified 35% early and 55% late apoptosis resulting from Que-MLs under the chemo-HT group. TEM results indicated MCF-7 cell membrane internalization and digestion of Que-MLs, suggesting the presence of early endosome-like vesicles on the cytoplasmic periphery. Conclusions Que-MLs exhibited multi-modal chemo-HT effects, displaying high toxicity against MCF-7 BC cells and showing promise as a potent cytotoxic agent for BC chemotherapy.

A versatile immunoassay based on functionalized nanoparticles for botulinum neurotoxin detection and sensor development

Botulinum neurotoxins (BoNTs) serotypes A and B are the two most common of the four BoNTs that cause the high mortality botulism disease in individuals consuming contaminated foods. The gold standard assay for BoNT detection is the live mouse bioassay, which has several major disadvantages, including tedious procedures and animal sacrifice requirements. In this study, we developed an immuno-based assay using magnetic streptavidin nanoparticles (mSNP) functionalized with specific synthetic biotinylated, 6xHis-tagged peptide substrates (Peptides A, PA and B, PB) designed for BoNT/A and BoNT/B proteolytic reactions, respectively. In the presence of active toxins that possess endopeptidase activity, upon cleavage, the released fragments with His-tag were dotted on a blotting membrane, ultimately producing color signals after incubation with anti-His antibody, alkaline phosphatase (AP)-conjugated antibody, and then AP substrates. The results showed that the efficiency of peptide-mSNP complex formation reached up to 81%, and the dot blot immunoassay allowed peptide detection from 10 ng of His-tagged peptides. Preliminary testing with the extracellular extracts from the isolated Clostridium botulinum strains indicated that the botulinum toxin in the 2020 botulism outbreak in Vietnam belonged to serotype A, the most potent BoNT. The established assay could be applied to construct a portable biosensor for BoNT detection and a high throughput device to screen potential BoNT inhibitors for drug development.

Expression of macrophage activation‑specific factors in hyperplastic scar tissue during hyperplasia phase by antibody array blotting membrane assay and its clinical significance.

The expression of macrophage activation-specific factors in hyperplastic scar (HS) tissues during hyperplasia phase was detected by antibody array imprinted membrane method and the role of macrophage activation in the natural evolution of HS was explored. A total of 83 patients with HS admitted to the Affiliated Hospital of Beihua University (Jilin, China) between February 2021 and July 2021 were enrolled. The clinical data of the patients were retrospectively analyzed. These patients were divided into the hyperplasia HS group (n=26) and the decline HS group (the HS tissues ceased to grow and were in regression periods; n=57) according to the time of scar formation and clinical characteristics. The HS tissues were collected from patients in both groups. The contents of IL-12, IL-10, VEGF and basic fibroblast growth factor (bFGF) were detected by antibody array imprinted membrane method and the contents of IL-12, IL-10, VEGF and bFGF in tissues with various groups of tissues and clinical features were compared. The connection between macrophage activation-specific factors with VEGF and bFGF was analyzed using Pearson correlation analysis. The contents of IL-10 (9.48±1.06), VEGF (24.15±2.64) and bFGF (37.48±2.56) were much lower and IL-12 levels (16.45±0.85) were strongly higher in hyperplasia HS group compared with those in the decline HS group (14.56±1.26 for IL-10, 27.85±2.63 for VEGF, 43.15±3.16 for bFGF and 10.46±0.75 for IL-12, P<0.001). In the hyperplasia HS group, the contents of IL-10, VEGF and bFGF were obviously higher and the IL-12 levels were markedly lower in patients with age ≥30 years, protuberance height <2 mm, soft flexibility, low hyperemia degree and no concomitant symptoms than those in the patients with age <30 years, protuberance height ≥2 mm, hard flexibility, high hyperemia degree and concomitant symptoms (P<0.001). Pearson correlation analysis showed that IL-12 was negatively correlated with VEGF and bFGF (r=-0.328, 0.600, P<0.01). IL-10 was positively correlated with VEGF and bFGF (r=0.486, 0.684, respectively, P<0.001). In conclusion, macrophage activation-specific factors were abnormally expressed in hyperplasia HS, mainly M1 macrophages, accompanied by severe inflammatory reaction. The transformation of M1 macrophage into M2 macrophage usually occurred during the declining HS phase, which accelerated scar formation by promoting the formation of fibroblasts and angiogenesis. Detection of macrophage activation-specific factors may contribute to evaluate the clinical stage of HS.

Evaluation of the Western Blot Densitometry for the Diagnosis of Congenital Toxoplasmosis

BACKGROUND: Congenital toxoplasmosis can cause severe ocular and neurological sequels such hydrocephaly, seizures, and chorioretinitis, leading to permanent visual deficit and multiple neurological deficits, if left untreated. An early diagnosis is crucial to prevent the development of these consequences and western blot is the most important diagnostic tool for this disease in newborns, because can distinguish between antibodies transferred from the mother or those produced by the newborn. However, the reading of the results still being done by subjective analysis by expert clinical laboratory specialists, reaching a maximum of 73% of sensitivity. The digitalization of the image of the results of western blot and its analysis by a software quantifying the densitometry of the bands obtained by western blot assay can potentially improve the diagnostic performance of the technique. METHODS: Was evaluated the sensitivity and specificity by digitalizing pictures of western blot results from the laboratory of Biomedical Research at the University of Quindío and published in the scientific literature. For the research, GelAnalyzer 19.1 software was used to digitalize the bands from western blot membranes from mother and child serum and the molecular weight of protein bands was calculated. The images were from 15 cases of newborns during the first month of life (six from our laboratory and nine from literature), and five negative controls (three from the laboratory and two from literature). Definition of true cases were the persistence of IgM/IgA after day 10 of life, mother with IgG and IgM antibodies positive and newborn with positive IgG plus symptoms, or no decrease in IgG in follow-up. The true negative cases were all newborn whose become negative for IgG anti Toxoplasma before the month 10 of life in absence of treatment, indicating that were maternal passively transferred antibodies. RESULTS: When comparing the immunological profiles of the mothers and their children for IgG by densitometry, congenital infections were detected in 80% (12 out of 15) of the cases (sensitivity) in serum samples obtained during the first month of life. All controls were negative (100% specificity). There was a percentage of 53% (8 out of 15) of children not identifiable with the visual analysis performed by two undergraduate researchers, likewise, the specificity with observation was 57% (3 out of 5) contrasted with the present study. Finally, three bands were identified as the most common in newborns with congenital infection: 49 KDa, 98-97 KDa and 72 KDa proteins (in order of frequency). CONCLUSION: Digitalization of western blot images increase sensitivity for the diagnosis of newborn with congenital toxoplasmosis during the first month of life.

Characterization of Linear IgE-Binding Epitopes in Food Allergens.

An IgE epitope is a part of an allergen that is capable of binding to IgE antibodies and eliciting an immune response. Identifying and characterizing human-allergy-relevant epitopes are important for diagnosis and prognosis of food allergy and development of immunotherapy treatments. This chapter describes the protocol for manual synthesis of overlapping peptides on a cellulose membrane and subsequent dot blotting of the peptides with allergic patients' IgE to map the linear IgE-binding epitopes in food allergens.

Enzymatically hydrolyzed fluorescence-based chemical probe enables in situ mapping of chitinase activity in the rhizosphere

Chitin is an insoluble and ubiquitous soil biopolymer, estimated to be the second most abundant organic soil biopolymer on Earth. Despite its abundance, role as a source of C and N in soil, and importance to ecosystem function, further research is required to elucidate key controls on chitin breakdown under varying environmental conditions. Previous work highlights the important role rhizosphere microbiomes and root exudates can play in chitin catabolism. To enable mapping of chitinase activity within the highly heterogeneous and spatially organized rhizosphere, we designed and synthesized an enzymatically activated fluorogenic substrate, chitotriose-TokyoGreen (chitotriose-TG), by incorporating a fluorescein derivative (TG) onto the trimeric unit of chitin. This non-fluorescent substrate is selectively hydrolyzed by chitinase to release TG and yield a fluorescence signal, which can be used to spatially image and measure chitinase activity in the rhizosphere. To demonstrate the application of this technique, we grew switchgrass (Panicum virgatum) in rhizoboxes amended with a horizontal layer supplemented with chitin. We extracted mobile proteins from the rhizobox using a nitrocellulose membrane blotting technique which offers non-destructive enzyme extraction while preserving the 2D spatial position of the enzymes. We then subjected these membranes to the synthesized chitotriose-TG stain to spatially visualize the distribution of chitinase activity within the rhizosphere. We observed increased chitinase activity near switchgrass roots and higher activity within the soil zone enriched in chitin, showing an adaptive response of chitinase production with spatial focusing in areas of higher chitin abundance. Thus, the enzyme extraction and visualization strategy we describe here can enhance efforts to better understand spatial controls on chitin breakdown in rhizosphere, further elucidating the role of chitin as a C and N source in these systems.

Heat-induced antigen retrieval utilizing modified Tris-EDTA buffer for reprobing of Western blots on nitrocellulose paper

The development of heat-induced antigen retrieval technologies with Tris-EDTA buffer has dramatically improved immunostaining of specific antigens for routine immunohistochemical detection (Krenacs et al., 2010) [1]. However, little evidence exists on whether heat-Induced antigen retrieval utilizing Tris-EDTA buffer can strip western blot (WB) membranes and allow sequential reprobing. Here, we serendipitously discover that ∼95 °C Tris-EDTA buffer with 0.01% Tween 20 could repeatedly strip the Nitrocellulose membranes (NC). After electroblotting, NC blots were soaked into Tris-EDTA stripping buffer (∼95 °C, 10–25min) and we could perform at least five rounds (the following antibodies used: Vinculin, Atg7, Caspase-3, UBA5, JNK and ERK1/2) stripping in sequential chemiluminescent detections. The NC membranes also show clear western signals and background without losing transferred proteins during the reprobing process of WB. Hence, this study report additional new roles of the heat-Induced antigen retrieval Tris-EDTA buffer with 0.01% Tween 20. The method is simpler, more affordable and harmless for the nitrocellulose paper, which will be helpful for effective reprobing in western blotting applications.

Expression, purification, characterization, and cytotoxic evaluation of the ML1-STxB fusion protein.

Targeted delivery of a toxin substance to cancer cells is one of the most recent cancer treatment options. Mistletoe Lectin-1 (ML1) in Viscum album L. is a Ribosome-inactivating proteins with anticancer properties. Therefore, it appears that a recombinant protein with selective permeability can be generated by fusing ML1 protein with Shiga toxin B, which can bind to Gb3 receptor that is abundantly expressed on cancer cells. In this study, we sought to produce and purify a fusion protein containing ML1 fused to STxB and evaluate its cytotoxic activities. The ML1-STxB fusion protein coding sequence was cloned into the pET28a plasmid, then was transformed into E. coli BL21-DE3 cells. Following induction of protein expression, Ni-NTA affinity chromatography was used to purify the protein. Using SDS-PAGE and western blotting, the expression and purification processes were validated. On the SkBr3 cell line, the cytotoxic effects of the recombinant proteins were evaluated. On SDS-PAGE and western blotting membrane, analysis of purified proteins revealed a band of approximately 41kDa for rML1-STxB. Ultimately, statistical analysis demonstrated that rML1-STxB exerted significant cytotoxic effects on SkBr3 cells at 18.09 and 22.52ng/L. The production, purification, and encapsulation of rML1-STxB fusion protein with potential cancer cell-specific toxicity were successful. However, additional research must be conducted on the cytotoxic effects of this fusion protein on other malignant cell lines and in vivo cancer models.

Trimethylamine-N-oxide sensitizes chondrocytes to mechanical loading through the upregulation of Piezo1.

Mechanical strain plays a crucial role in chondrocyte apoptosis and osteoarthritis (OA) disease progression through Piezo1. Trimethylamine-N-oxide (TMAO) is a diet-derived metabolite that correlates positively with multiple chronic diseases. Herein, we explored the potential role of TMAO in sensitizing chondrocytes to Piezo1-mediated mechanotransduction. The cytotoxicity of TMAO on chondrocytes was assayed. Piezo1 expression was measured after TMAO intervention. Pathological mechanical loading or Yoda1 (a specific Piezo1 channel activator) was administered in chondrocytes. The calcium levels and cytoskeleton in chondrocytes were observed by fluorescence microscopy. Flow cytometry, western blotting, and mitochondrial membrane potential assays were utilized to evaluate apoptosis. A rat OA model was constructed by anterior cruciate ligament transection. Hematoxylin-eosin staining, Safranin-O/Fast Green staining, immunochemistry, and TUNEL were applied to estimate OA severity. TMAO intervention alone did not affect chondrocyte viability up to 600μM. TMAO significantly increased Piezo1 expression and up-regulated intracellular calcium levels, further leading to cytoskeletal damage. Mechanical strain or Yoda1 treatment significantly induced chondrocyte apoptosis. Notably, TMAO intervention further aggravated chondrocyte apoptosis and cartilage destruction under pathological mechanical loading. TMAO significantly up-regulated Piezo1 expression and sensitized chondrocytes to mechanical loading, which may be closely related to the pathogenesis of OA.

Celecoxib activates autophagy by inhibiting the mTOR signaling pathway and prevents apoptosis in nucleus pulposus cells

BackgroundIntervertebral disc degeneration results from a variety of etiologies, including inflammation and aging. Degenerated intervertebral discs feature down-regulated extracellular matrix synthesis, resulting in losing their ability to retain water and absorb compression. Celecoxib is a well-known selective cyclooxygenase-2 inhibitor for treating arthritis and relieving pain. Nevertheless, the mechanism of Celecoxib for treating inflammation-related intervertebral disc degeneration has not yet been clarified.MethodProtein synthesis was analyzed by western blot. Fluorescent probes DCFH-DA and MitoSox Red detected reactive oxygen species and were measured by flow cytometry. The activity of the kinase pathway was evaluated by protein phosphorylation. Autophagy was monitored by mRFP-GFP-LC3 transfection and LC3 analysis. Mitochondrial apoptotic proteins were analyzed by western blot and cell membrane integrity was measured by flow cytometry. The autophagic gene was silenced by siRNA.ResultsIn this study, interleukin-1β stimulation reduced the synthesis of aggrecan, type I and II collagen and caused excessive production of reactive oxygen species. We looked for a therapeutic window of Celecoxib for nucleus pulposus cells to regain extracellular matrix synthesis and reduce oxidative stress. To look into nucleus pulposus cells in response to stimuli, enhancement of autophagy was achieved by Celecoxib, confirmed by mRFP-GFP-LC3 transfection and LC3 analysis. The mammalian target of rapamycin and a panel of downstream proteins responded to Celecoxib and propelled autophagy machinery to stabilize homeostasis. Ultimately, inhibition of autophagy by silencing autophagy protein 5 disrupted the protective effects of Celecoxib, culminating in apoptosis.ConclusionIn summary, we have demonstrated a new use for the old drug Celecoxib that treats intervertebral disc degeneration by enhancing autophagy in nucleus pulposus cells and opening a door for treating other degenerative diseases.

Determining Potency of Inhibitors Targeting Histone Deacetylase 6 by Quantification of Acetylated Tubulin in Cells.

During the preclinical development of small molecule inhibitors, compounds or compound libraries are typically first screened using purified target enzymes in vitro to select candidates with high potency. In the later stages of the development, however, functional cell-based assays may provide biologically more relevant data. In this chapter, we describe a detailed protocol for determining the potency of inhibitors targeting human histone deacetylase 6 in complex cellular environments. Cells are first treated with a dilution series of tested compounds, cell lysates separated by SDS-PAGE, and electrotransferred to a blotting membrane. The inhibitor potency is then determined indirectly by quantifying the levels of acetylated tubulin as a surrogate readout.

Stain-Free total-protein normalization enhances the reproducibility of Western blot data

We compared the accuracy of three common methods of total protein normalization. The Stain-Free method was accurate across different types/brands of western blotting membrane and for various protein loads, unlike Ponceau S and Amido Black. Normalizing to the housekeeping proteins Actin and β-Tubulin could match the accuracy of the Stain-Free method. However, compared to Actin or β-Tubulin, normalizing to the Stain-Free signal reduced variability that led to enhanced reproducibility and a reduction in the number of samples needed to obtain statistically significant results by >50%. Stain-Free normalization can enhance the reproducibility and hence the confidence in Western Blot data.

P–580 Levels of the constitutive heterochromatin (cHC) mark H3K9me3 in human sperm in relation to IVF outcome and preimplantation embryo development

Abstract Study question Can we quantify the variation in epigenetic signature of constitutive heterochromatin (cHC) in human sperm using Western blot analysis and correlate this with pregnancy outcome after IVF? Summary answer Variation in the quantified H3K9me3 to H3 showed an association with embryo morphokinetics and a H3k9me3/H3 ratio &amp;lt;0,5 is negatively associated with pregnancy outcome. What is known already In human sperm, 5–15% of the DNA remains associated to histones. We previously showed that human mature spermatozoa still exhibit the canonical marks H3K9me3 and H4K20me3 on cHC regions and transmit these nucleosomes to the embryo after fertilization. Additionally, other groups reported an increase in the nucleosome/protamine ratio when sperm from male factor subfertility patients were compared with sperm from fertile men, indicating that incomplete chromatin remodelling during spermatid elongation may affect fertility. Thus, variations in sperm chromatin occur during human spermatogenesis that go undetected by current sperm quality tests and may have clinical significance for IVF treatment outcomes. Study design, size, duration An observational study of patient couples (n = 53) undergoing IVF treatment at the Erasmus MC, Rotterdam, between 2017 and 2018. Inclusion criteria were normospermia according to the WHO criteria, at least 4 oocytes harvested after ovum pickup and an embryo-transfer. After routine insemination, resulting embryos from 38 cycles were cultured in the EmbryoScopeTM and developmental timings up to the 8-cell stage were recorded. The primary outcome measure was ongoing pregnancy (ultrasound at 12 weeks of gestation). Participants/materials, setting, methods Surplus sperm samples from 53 IVF cycles were lysed at a concentration of 200x106 spermatozoa/ml in RIPA buffer. Lysates were loaded and separated on SDS-PAGE gels and transferred onto blotting membranes. After incubation with antibodies against H3 and H3K9me3 and fluorescent detection, signal intensity was quantified using Image Studio Lite (LI-COR Biotechnology)). Additionally, time-lapse developmental kinetics of 130 transferred and cryopreserved embryos of 38 patient couples were analyzed. Main results and the role of chance No significant differences were found in patient characteristics between the pregnant and non-pregnant group. However, fertilization rate was found to be a confounding factor; no pregnancies occurred below a fertilization rate of 40% and only 4 occurred below a fertilization rate of 60%. The median H3k9me3/H3 ratio in the pregnant group was 1.01 (interquartile range (IQR): 0.69–1.16) and in the non-pregnant group 0.896 (IQR: 0.54–1.06), this difference was not significant. When using logistic regression analysis, adjusted for the fertilization rate (dichotomized as ≤ 60% or &amp;gt; 60%), we observed a positive association between the H3k9me3/H3 ratio and ongoing pregnancy (odds ratio (OR) 6.43 (95% confidence interval (CI): 1.12–36.8; p-value 0.037). More importantly, none of the samples with a ratio of 0.5 or lower resulted in an ongoing pregnancy (n = 7). After classifying sperm samples into quartiles according to the H3K9me3/H3 ratio, we observed that resulting embryos reached the 4-cell stage faster with sperm from to the lowest quartile of H3K9me3/H3 ratio, compared to the highest quartile (beta –4.4 hours, p-value 0.05). Our results point to an association between the H3k9me3/H3 ratio, embryo development and pregnancy outcome. Limitations, reasons for caution This initial analysis is performed with a relatively low number of samples. The OR of 6.43 has a wide 95% CI, probably due to low statistical power. Our observations await confirmation in a larger data set. Wider implications of the findings: Semen analysis remains the cornerstone of male infertility diagnosis, despite its low prognostic value. Sperm epigenetic cHC inheritance likely has an important impact on early embryo development. Therefore, the H3k9me3/H3 ratio could serve as a parameter for sperm quality and aid in the prediction of pregnancy outcome. Trial registration number Not applicable

FC 014INFLUENCE OF GENETIC VARIATION IN SLC7A13/AGT1 IN HUMAN CYSTINURIA

Abstract Background and Aims Cystinuria (CU) is an inherited renal disorder based on urinary wasting of dibasic amino acids, urinary precipitation, and consecutive cystine stone formation. It is caused by pathogenic variants in two distinct disease genes, SLC3A1 and SLC7A9, both of which encode subunits of a heterodimeric tubular amino acid transporter, rBAT/SLC3A1 and BAT1/SLC7A9, located at the apical membrane of proximal renal tubules. CU is marked by incomplete penetrance and substantial disease variability. Recently, a novel cystine transporter, consisting of the light chain AGT1/SLC7A13 and its heterodimeric partner rBAT/SLC3A1 has been identified in the S3 segment of murine proximal tubules. In this study, we aim at evaluating the role of AGT1 in cystinuric patients with or without mutations in either SLC3A1 or SLC7A9, analyzing the role of AGT1/SLC7A13 as novel disease gene or genetic modifier in CU. Method A multicenter European CU-cohort comprising 132 individuals was screened for pathogenic variants in SLC3A1, SLC7A9, and SLC7A13 using high-throughput multiplex PCR-based amplification and next-generation sequencing (MiSeq Illumina) followed by multiplex ligation-dependent probe amplification (MLPA) of SLC3A1 and SLC7A9. For functional in vitro studies, epitope-tagged human and murine rBAT and AGT1 proteins were transiently expressed in different cell systems. Heterodimer complex formation was analyzed by co-immunoprecipitation and western blot studies and membrane trafficking was evaluated by immunofluorescence microscopy. Results Genectic analysis of our CU-cohort did not reveal indiviuals with SLC7A13 variation only, however we found three patients harbouring heterozygous missense variants in addition to pathogenic or VUS variants in SLC3A1 or SLC7A9. To evaluate their influence on the generation of functional cystine transporters in vitro, different cell models were transiently transfected with plasmids expressing wildtype or mutant proteins. In line with previous reports, co-expression of AGT1 and rBAT wildtype allowed efficient complex formation as AGT1-induced maturation of rBAT was detected by increased mature N-glycosylation, co-immunoprecipitation and membrane insertion. Whereas AGT1 patient variants p.Met452Thr (SLC7A13 c.1355T&amp;gt;C) and p.Ile174Phe (SLC7A13 c.520A&amp;gt;T) behaved comparable to wildtype AGT1, variants p.Asn45Lys (SLC7A13 c.135C&amp;gt;G) and p.Leu270Phe (SLC7A13 c.808C&amp;gt;T) led to clearly reduced glycosylation patterns and trafficking deficits of rBAT wildtype protein. Next, the mutual influence of pathogenic variation in both, AGT1 and rBAT, will unravel the consequences of patient-specific molecular interactions on the functional expression of cystine transporter complexes. Conclusion Here, we report three CU-patients with variants in SLC7A13 combined with either SLC3A1 or SLC7A9. For two of these variants, in vitro functional analysis revealed pathogenic molecular mechanisms disturbing complex formation, maturation and trafficking of rBAT. We hypothesize that specific pathogenic variants in SLC7A13 interfere with efficient membrane localization of heterodimeric cystine transporters, which results in modulation of cystine transport in the S3 segment of proximal tubules in CU-patients.

Spreader-CDR system for efficient, reproducible, and frugal western blot.

Efficient antibody incubation is a vital step for successful western blot. During the incubation, a thin antibody-depleted layer is created around the blotting membrane, which limits antibody binding. Although the conventional batch shaking method is ineffective against it, this layer can be easily disrupted by cyclic draining and replenishing (CDR) of the antibody solution during membrane incubation. Previously, we introduced a closed and rotating cylindrical chamber as a tool to implement CDR for western blots (rCDR). A new open bucket-style chamber was devised for easier operation and the possibility of process automation. Instead of rotation as in rCDR, rocking it back and forth achieved the CDR antibody incubation (R-CDR). The chamber was then equipped with a spreader-rod to facilitate the uniform movement of the antibody solution across the membrane surface. Hence, it was named spreader CDR (S-CDR). Compared to the batch incubation method, both the S-CDR and R-CDR devices produced significantly enhanced signals and developed faster results. There were several additional benefits of using the spreader-rod, which included uniform antibody binding across the membrane, reduced usage of antibodies, and the ability to recover results even from mishandled, creased membranes. The S-CDR device ensures better blots and can be easily implemented in existing western blot protocols.

Gold-Silver and Gold-Palladium Alloy Nanoparticles as Mass-Probes for Immunosensing.

Silver or palladium shelled gold nanoparticles were fused into alloy nanoparticles by pulsed-laser irradiation. The alloy nanoparticles could carry antibodies on their surfaces without affecting their immune functionalities and interact selectively with antigens on a blotting membrane. Silver or palladium ions desorbed from the alloy nanoparticles as reporter ions upon the UV laser irradiation in a mass spectrometer.

Monoclonal antibody against Nile tilapia (Oreochromis niloticus) IgM heavy chain: A valuable tool for detection and quantification of IgM and IgM+ cells

Nile tilapia (Oreochromis niloticus) is a freshwater fish, which is extensively cultivated worldwide and constitutes one of the model species for the study of fish immunology. Monoclonal antibodies are very advantageous molecular tools for studying teleost immune system. Specifically, monoclonal antibodies that react with immunoglobulins are used successfully in the study of the humoral immune response of several fish species. In the present study, we produced and characterized a monoclonal antibody against tilapia IgM heavy chain using a peptide-based strategy. The peptide sequence was selected from the surface-exposed region between CH3–CH4 domains. The specificity of the polyclonal serum and the hybridoma culture supernatant obtained by immunization with the peptide conjugated to keyhole limpet hemocyanin were evaluated by western blotting, both showing reactivity against tilapia serum IgM. The purified mAb was able to recognize secreted IgM by western blotting and ELISA and membrane IgM by flow cytometry. We also demonstrated that the antibody doesn't cross-react with a recombinant IgT fragment. This tool allowed us to study for the first time the stimulation of mucosal immunity after Pituitary Adenylate Cyclase Activating Polypeptide administration. Overall, the results demonstrated the utility of this mAb to characterize humoral immune response in O. niloticus.

Semi-automated analysis of dot blots using ImageJ/Fiji

Commercially available dot blots provide a set of specific antibodies spotted on membranes in a given pattern. If the target analyte is present in the solution that the membrane is incubated with, the detection reaction will result in a chemiluminescence signal which is recorded by film or scanner. In order to know which analytes were detected, the analysis consists of measuring the intensity of the recorded signal on each spot. Manually measuring the entire array (typically ~200 spots) is unreliable and tedious. Fully automatic registration of the blot membrane to the template pattern often fails since there might be only very few positive spots on the membrane. This article presents an ImageJ/Fiji macro that requires minimal user input to perform a robust iterative registration of an adjustable template mask representing the spot pattern to the recorded blot. Once the template mask is matched to the dot blot, the spot intensity of each dot is measured and reported in a results table.

Unlocking the brain: A new method for Western blot protein detection from fixed brain tissue

BackgroundAldehyde fixation is a common process used to preserve the complex structure of biological samples ex vivo. This method of fixation relies on the formation of covalent bonds between aldehydes and amines present in the biomolecules of the sample. Aldehyde fixation is routinely performed in histological studies, however fixed tissue samples are rarely used for non-histological purposes as the fixation process is thought to make brain tissue unsuitable for traditional proteomic analyses such as Western blot. Advances in antigen-retrieval procedures have allowed detectable levels of protein to be solubilized from formaldehyde fixed tissue, opening the door for aldehyde-fixed samples to be used in both histological and proteomic approaches. New methodHere, we developed a series of antigen-retrieval steps for use on fixed-brain lysates to make them suitable for analysis by Western blot. ResultsProlonged exposure of the tissue homogenate to high temperature (90 °C for 2 h) in the presence of a concentrated formaldehyde scavenger and ionic detergent was sufficient to reveal a variety of synaptic and non-synaptic proteins on membrane blots. ConclusionThis protocol has significant utility for future studies using fixed tissue samples in a variety of neuropathological conditions.