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Proteins, saccharides, and low molecular organic compounds in the blood, urine, and saliva could potentially serve as biomarkers for diseases related to diet, lifestyle, and the use of illegal drugs. Lifestyle-related diseases (LSRDs) such as diabetes mellitus (DM), non-alcoholic steatohepatitis, cardiovascular disease, hypertension, kidney disease, and osteoporosis could develop into life-threatening conditions. Therefore, there is an urgent need to develop biomarkers for their early diagnosis. Advanced glycation end-products (AGEs) are associated with LSRDs and may induce/promote LSRDs. The presence of AGEs in body fluids could represent a biomarker of LSRDs. Urine samples could potentially be used for detecting AGEs, as urine collection is convenient and non-invasive. However, the detection and identification of AGE-modified proteins in the urine could be challenging, as their concentrations in the urine might be extremely low. To address this issue, we propose a new analytical approach. This strategy employs a method previously introduced by us, which combines slot blotting, our unique lysis buffer named Takata's lysis buffer, and a polyvinylidene difluoride membrane, in conjunction with electrospray ionization-mass spectrometry (ESI)/matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). This novel strategy could be used to detect AGE-modified proteins, AGE-modified peptides, and free-type AGEs in urine samples.

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Advanced glycation end products (AGEs) are formed through the reaction/modification of proteins by saccharides (e.g., glucose and fructose) and their intermediate/non-enzymatic products [e.g., methylglyoxal and glyceraldehyde (GA)]. In 2017, Dr. Takanobu Takata et al. developed the novel slot blot method to quantify intracellular GA-derived AGEs (GA-AGEs). Although the original method required nitrocellulose membranes, we hypothesized that the modified proteins contained in the AGEs may be effectively probed on polyvinylidene difluoride (PVDF) membranes. Because commercial lysis buffers are unsuitable for this purpose, Dr. Takata developed the slot blot method using an in-house-prepared lysis buffer containing 2-amino-2-hydromethyl-1,3-propanediol (Tris), urea, thiourea, and 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) that effectively probes AGEs onto PVDF membranes. The slot blot method also entails the calculation of Tris, urea, thiourea, and CHAPS concentrations, as well as protein and mass to be probed onto the PVDF membranes. GA-AGE-modified bovine serum albumin (BSA, GA-AGEs-BSA) is used to draw a standard curve and perform neutralization against a non-specific combination of anti-GA-AGEs antibodies, thereby enabling the quantification of GA-AGEs in cell lysates. This paper presents the detailed protocol for slot blot analysis of intracellular GA-AGE levels in C2C12 cells. Key features • This protocol leverages the idea that advanced glycation end products are modified proteins. • The lysis buffer containing Tris, urea, thiourea, and CHAPS enables probing proteins onto PVDF membranes. • Intracellular GA-AGE levels may be quantified for some cell types using polyclonal anti-GA-AGE antibodies and standard GA-AGE-modified BSA. • The lysis buffer may be simultaneously prepared with the cell lysate. • There is no limit to the type of cultured cells used in the preparation of cell lysate.

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Kampo is a Japanese traditional medicine modified from traditional Chinese medicine. Kampo medicines contain various traditional crude drugs with unknown compositions due to the presence of low-molecular-weight compounds and proteins. However, the proteins are generally rare and extracted with high-polarity solvents such as water, making their identification and quantification difficult. To develop methods for identifying and quantifying the proteins in Kampo medicines, in the current study we employ previous technology (e.g., column chromatography, electrophoresis, and membrane chromatography), focusing on membrane chromatography with a polyvinylidene difluoride (PVDF) membrane. Moreover, we consider slot blot analysis based on the principle of membrane chromatography, which is beneficial for analyzing the proteins in Kampo medicines as the volume of the samples is not limited. In this article, we assess a novel slot blot method developed in 2017 and using a PVDF membrane and special lysis buffer to quantify advanced glycation end products-modified proteins against other slot blots. We consider our slot blot analysis superior for identifying and quantifying proteins in Kampo medicines compared with other methods as the data obtained with our novel slot blot can be shown with both error bars and the statistically significant difference, and our operation step is simpler than those of other methods.

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DNA-protein crosslinks (DPCs) are steric hindrances to DNA metabolic processes and the removal and repair of DPCs is a rapidly evolving area of research. A critical component of deciphering this repair pathway is developing techniques that detect and quantify specific types of DPCs in cells. Here we describe a protocol for direct detection of enzymatic DPCs from mammalian cells-the RADAR assay. The method involves isolating genomic DNA and DPCs from cells and binding them to nitrocellulose membrane with a vacuum slot blot manifold. DPCs are detected using antibodies raised against the protein of interest and quantified by normalizing to a DNA loading control. The RADAR assay allows for the detection of specific types of DPCs and the sensitive analysis of the DNA-protein crosslinking activity of various drugs, is adaptable across different cell types and conditions, and requires little specialized equipment.

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Various types of advanced glycation end-products (AGEs) have been identified and studied. I have reported a novel slot blot analysis to quantify two types of AGEs, glyceraldehyde-derived AGEs, also called toxic AGEs (TAGE), and 1,5-anhydro-D-fructose AGEs. The traditional slot blot method has been used for the detection and quantification of RNA, DNA, and proteins since around 1980 and is one of the more commonly used analog technologies to date. However, the novel slot blot analysis has been used to quantify AGEs from 2017 to 2022. Its characteristics include (i) use of a lysis buffer containing tris-(hydroxymethyl)-aminomethane, urea, thiourea, and 3-[3-(cholamidopropyl)-dimetyl-ammonio]-1-propane sulfonate (a lysis buffer with a composition similar to that used in two-dimensional gel electrophoresis-based proteomics analysis); (ii) probing of AGE-modified bovine serum albumin (e.g., standard AGE aliquots); and (iii) use of polyvinylidene difluoride membranes. In this review, the previously used quantification methods of slot blot, western blot, immunostaining, enzyme-linked immunosorbent assay, gas chromatography-mass spectrometry (MS), matrix-associated laser desorption/ionization-MS, and liquid chromatography-electrospray ionization-MS are described. Lastly, the advantages and disadvantages of the novel slot blot compared to the above methods are discussed.

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DNA topoisomerases resolve topological stress by introducing transient single- or double-strand breaks into the DNA duplex. This reaction requires the covalent binding of topoisomerases to DNA while the topological stress is being released. This transient intermediate is known as topoisomerase-covalent complex and represents the target of many anti-cancer drugs. Here, we describe a protocol to quantitatively detect topoisomerase-covalent complexes in vivo, called RADAR (rapid approach to DNA adduct recovery). DNA and protein-DNA covalent complexes are rapidly isolated from cells through chaotropic extraction. After normalization, samples are loaded on a slot blot, and the covalent complexes are detected using specific topoisomerase antibodies. In addition to being fast and robust, this assay produces quantitative results. Consequently, the RADAR assay can be applied to investigate the topoisomerase-covalent complex biology, including the effect of specific topoisomerase inhibitors. Finally, the same assay can be more generally applied to study covalent complexes of other enzymes with DNA.

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R-loops are non-B-DNA structures consisting of an RNA/DNA hybrid and a displaced single-stranded DNA. They arise during transcription and play important biological roles. However, perturbation of R-loop levels represents a source of DNA damage and genome instability resulting in human diseases, including cancer and neurodegeneration. In this chapter, we describe a protocol which allows detection of R-loop interactors using affinity purification with S9.6 antibody, specific for RNA/DNA hybrids, followed by Western blotting or mass spectrometry. Multiple specificity controls including addition of synthetic competitors and RNase H treatment are described to verify the specificity of identified R-loop-binding factors. The identification of new R-loop interacting factors and the characterization of their involvement in R-loop biology provides a powerful resource to study the role of these important structures in health and disease.

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Pru p 3, one of the most representative proteins of the lipid transfer proteins (LTPs), is responsible for clinical allergic reactions to food of peach origin. The identification of Pru p 3 epitopes is not comprehensive due to different methods and principles of epitope screening. In addition, evaluation of the stability of the epitopes and the validation of the immunological key amino acids still need further research. Therefore, in the present study, an immune slot-blot microarray assay was performed to screen the epitopes from Pru p 3 overlapping peptide library, and a new epitope (P-1, AA1-16, ITCGQVSSALAPCIPY) was identified and two identified epitopes were deeply investigated (P-2, AA12-27, PCIPYVRGGGAVPPAC; P-3, AA23-38, VPPACCNGIRNVNNLA). The stability of these epitopes was then verified by thermal processing treatment and digestion experiments. Moreover, the key amino acids of the three identified epitopes were obtained by epitope amino acid mutation combined with slot-blot experiments. These findings may contribute to the further understanding of Pru p 3 and the prevention of peach allergy.

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Beta-thalassemia is the most frequent hemoglobin disorder in Iran resulting from disrupting mutations in the beta globin (HBB) gene that causes decreased or complete absent of beta-globin chains. The screening of beta-thalassemia minor and major individuals and prenatal diagnosis is important for familial planning. Therefore, it is essential, depending on the ethnicity and local frequency of changes, to develop a rapid and accurate method for molecular diagnosis of beta-thalassemia. Here, we developed reverse slot blot (RSB) assay for the simultaneous detection of six common pathogenic changes in the HBB gene (-88, -28, IVSII-745, IVSII-848, Codon 6 [G → A] for HbC, Codon 6 [A → T] for HbS) in the Khuzestan Province of Iran. We designed normal and mutant oligonucleotide probes for each selected mutation and fixed them on positively charged nylon membrane. In the next step, a multiplex-polymerase chain reaction (PCR) performed for the amplification of the entire HBB gene using labelled 5'-biotinylated primers. The PCR products were hybridized to immobilized oligonucleotide probes on the membrane at the appropriate temperature. Finally, we developed the membrane by chemically colorimetric reaction using nitro-blue tetrazolium-5-Bromo-4-chloro-3-indolyl phosphate. For the best probe concentration, we made a serial dilution of probe pairs for each mutation. The optimal probe concentration for each mutation varied from 25 to 50 pmol. In the next step, DNA samples from homozygous affecting individuals were subjected for multiple PCR. Hybridization of each PCR products on the nylon membrane with probe pairs revealed specific bands with expected signal intensity without any background. Our designed RSB test is a rapid, sensitive and cost-effective method for screening of regional specific beta-thalassemia mutations in the Khuzestan population of Iran, which might be extended for the detection of any desired pathogenic changes.

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Chromatin immunoprecipitation (ChIP) is a versatile method to investigate the interaction between specific proteins and DNA regions in vivo. The success of ChIP experiments is highly dependent on the quality of chromatin preparation and especially DNA fragmentation. To ascertain whether DNA fragmentation is appropriate for ChIP experiments, agarose gel electrophoresis is required. Here we describe the experimental procedure of agarose gel electrophoresis to verify whether DNA fragmentation is appropriate for ChIP-slot blot experiments.

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Clostridium septicum produces a number of diseases in human and farm animals which, in most of the cases, are fatal without clinical intervention. Alpha toxin is an important agent and the unique lethal virulent factor produced by Clostridium septicum. This toxin is haemolytic, highly lethal and necrotizing activities but is being used as an antigen to develop animal vaccines. The aim of this study was to isolate the alpha toxin of Clostridium septicum and produce highly specific antibodies against it. In this work, we have developed a simple and efficient method for alpha toxin purification, based on electroelution that can be used as a time-saving method for purifying proteins. This technique avoids contamination by other proteins that could appear during other protein purification techniques such chromatography. The highly purified toxin was used to produce polyclonal antibodies. The specificity of the antibodies was tested by western blot and these antibodies can be applied to the quantitative determination of alpha toxin by slot blot.

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Studies of the interaction of bacteria with mucus-secreting cells can be complemented at a more mechanistic level by exploring the interaction of bacteria with purified mucins. Here we describe a far Western blotting approach to show how C. jejuni proteins separated by SDS PAGE and transferred to a membrane or slot blotted directly onto a membrane can be probed using biotinylated mucin. In addition we describe the use of novel mucin microarrays to assess bacterial interactions with mucins in a high-throughput manner.

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Pollen aeroallergens are present in atmospheric particulate matter (PM) where they can be found in coarse biological particles such as pollen grains (aerodynamic diameter dae>10µm), as well as fragments in the finest respirable particles (PM2.5; dae<2.5µm). Nitration of tyrosine residues in pollen allergenic proteins can occur in polluted air, and inhalation and deposition of these nitrated proteins in the human respiratory tract may lead to adverse health effects by enhancing the allergic response in population. Previous studies investigated protein nitration by atmospheric gaseous pollutants such as nitrogen dioxide and ozone. In this work we report, for the first time, a study on protein nitration by nitrate ion in aqueous solution, at nitrate concentrations and pH conditions simulating those occurring in the atmospheric aerosol liquid water phase. Experiments have been carried out on the Bovine serum albumin (BSA) protein and the recombinant Phleum pratense allergen (Phl p 2) both in the dark and under UV-A irradiation (range 4-90Wm-2) to take into account thermal and/or photochemical nitration processes. For the latter protein, modifications in the allergic response after treatment with nitrate solutions have been evaluated by immunoblot analyses using sera from grass-allergic patients. Experimental results in bulk solutions showed that protein nitration in the dark occurs only in dilute nitrate solutions and under very acidic conditions (pH<3 for BSA; pH<2.2 for Phl p 2), while nitration is always observed (at pH0.5-5) under UV-A irradiation, both in dilute and concentrated nitrate solutions, being significantly enhanced at the lowest pH values. In some cases, protein nitration resulted in an increase of the allergic response.

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BACKGROUND/AIM: Accumulating evidence shows that various types of cancers induce a specific immune response resulting in the production of antibodies against self-components (autoantibodies). The aim of the present study was to identify antigens for autoantibodies in sera from patients with ovarian cancer, especially clear cell carcinoma (CCC), as novel diagnostic markers for the disease. MATERIALS AND METHODS: The reactivity of individual sera from patients was examined by two-dimensional (2-D) immunoblotting using lysates of CCC cell lines, ES-2 and RMG-1, as antigens to identify autoantigens. ELISA was established to quantitatively measure autoantibody titer of patients' sera. RESULTS: Autoantibodies against RhoGDI were induced in sera of ovarian cancer patients. Elevated levels of autoantibodies against heterogeneous nuclear ribonucleoprotein L (hnRNPL) and a mitochondrial protein, dihydrolipoamide dehydrogenase (DLD), were detected in patients with CCC. CONCLUSION: Autoantibodies against RhoGDI and hnRNPL and DLD may serve as novel diagnostic markers for ovarian cancer and CCC, respectively.

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Kindling development is a good animal model of epilepsy and neural plasticity. It is induced by repeated subconvulsive electrical or chemical stimulations. This leads to progressive and permanent amplification of seizure activity resulting in permanent brain changes. Immediate early genes(IEGs) are proposed as the master switch for turning on molecular events in long term neural plasticity. The role of c-myc, an IEG, in the development of kindling is not known. This study was conducted to investigate the role of c-myc in the neural plastic changes underlying kinding. Among 115 adult male Spargue-Dawley rats, 51 were kindled by repeated administrations of subconvulsive doses(15-25mg/kg) of pentylenetetrazol(PTZ). Twenty-eight rats experienced various degree of convulsions induced by a single injection of convulsive dose(30-60mg/kg) of PTZ. Eighteen rats experienced mild or severe convulsions induced by a single injection of convulsive dose(30-60mg/kg) of PTZ. Eighteen rats experienced mild or severe convulsion by a single electroconvulsive shock(ECS). Eighteen rats received normal saline as a control group. Animals were sacrificed in 30 minutes, 1 hour and 48 hours after convulsion. C-myc mRNA levels in the hippocampus were quantified using slot-blot hybridization analysis. In the experiment of PTZ kindling, c-myc mRNA expression 30 minutes after convulsion was elevated about 3-8 times compared with controls. C-myc mRNA expression 1 hour after convulsion was elevated about 4 times at stage I, II, and V, ut was not elevated at stage III and IV. C-myc mRNA expression 48 hours after convulsion was elevated about 2-3 times compared with controls. In the experiment of PTZ-induced seizures, c-myc mRNA expression 30 minutes after convulsion was elevated 5-6 times compared with controls. C-myc mRNA expression 1 hour after convulsion was elevated 4-6 times. C-myc mRNA expression 48 hours after convulsion was elevated approximately 2 times. In the experiment of ECS-induced seizures, c-myc mRNA expression was elevated 4 times at 30 minutes and 1 hour after mild convulsion, but decreased at 30 minutes and 1 hour after severe convulsion compared with control. C-myc mRNA expression 48 hours after convulsion was elevated approximately 2 times. These results suggest that the enhanced expression of c-myc mRNA is a non-specific consequence in the development of PTZ kindling. In addition, c-myc does not seem to play an important role in turning on a molecular program underlying kindling.

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