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Western Blot - Crossref

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Last Updated: 09 January 2023

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The answer depends on the question: Optimal conditions for western blot characterization of muscle collagen type 1 depends on desired isoform

Fibrillar collagen type 1 is the most abundant form of collagen in the body and is a vital component of extracellular infrastructure. To detect changes in procollagen versus mature collagen at the protein level, improved techniques are needed in order to assess collagen production and extracellular accumulation in fibrotic disorders. The analysis shows that full native gel conditions prevent the absorption of all collagen type 1 bands. Type 1 procollagen gels are obtained using 4% to 12% Tris-glycine gels without the presence of SDS in the gel itself, but SDS in the running and sample buffers are required. Both 8% and gradients type gels are suitable for analysis of mature collagen 1, however, there are still without SDS, but with SDS included in both running and sample buffers, BME must be added to the sample buffer, and samples should not be boiled.

Source link: https://doi.org/10.14440/jbm.2019.289


Anti-RAINBOW dye-specific antibodies as universal tools for the visualization of prestained protein molecular weight markers in Western blot analysis

Abstract: Western blotting is one of the most commonly used methods in molecular biology and biochemistry. In protein electrophoresis, pre-prepared proteins are used as molecular weight standards. These colored protein markers are not visible in the chemiluminescent Western blot review, but they leave researchers with the sad situation that the protein of interest and the marker's signal are not captured simultaneously and must be merged in an error-prone step. We created monoclonal antibodies specific for the protein dyes to enable simultaneous detection of marker proteins. In which a new carrier protein was used for each subsequent immunization, we immunized mice sequentially with dye-carrier protein complexes in which a new carrier protein was used to elicit a dye rather than a protein specific immune response. Our novel antibodies are useful for simultaneous Western blot analysis of commercially available prestained marker proteins in conjunction with the characterization of any specific protein of concern. Our novel antibodies are convenient devices for simultaneous Western blot detection of commercially available prestained marker proteins in combination with the detecting of any particular protein of concern.

Source link: https://doi.org/10.1038/srep31363


Diagnostic Performance of Competitive ELISA and Western Blot Methods for the Detection of Antibodies against Theileria equi and Babesia caballi

Equine piroplasmosis is the causative pathogens of Equine piroplasmosis, a disease that has resulted in major economic losses and severe restrictions to the global equine industry. We optimized the dilution of T. equi or B. caballi positive serum samples to 1:200 in the newly developed western blot assays. Both the novel cELISA assay and the western blot assay were found to have high correlation rates in antibodies testing T. equi and B. caballi, relative to the commercially available kit. The novel cELISA and the western blot assays for antibodies against T. equi or B. caballi may be able to quickly test for T. equi or B. caballi, as well as contributing to the detection and control of the disease.

Source link: https://doi.org/10.3390/microorganisms11010021


Undergraduate immunology lab project including opportunities for experimental design, a poster presentation, and the opportunity to partially purify antibodies from hybridoma cells to utilize in a Western blot

Abstract Undergraduate students benefit from laboratory experiments that more closely mimic research findings. Students maintain the hybridoma cell line MARC 2B7 and design a mini-research project to investigate antibody production under a modified growth condition of their choice during this 7-week lab project. The students are given a common lab protocol as a starter and a list of revisions and/or changes so that the students are responsible for creating their own final protocol for each phase. For example, the dot blot protocol is based on chemiluminescent detection; however, HRP conjugated means that students are also supplied with the manufacturer's information on the secondary antibody and the HRP substrate, which can be obtained in lab. The antibodies from hybridoma supernatants that had promising results on the dot blots are also used by lab groups in the aftermath.

Source link: https://doi.org/10.4049/jimmunol.204.supp.222.12


Serologic, biologic and western blot analysis of human IgE-binding factor derived from a T cell hybridoma maintained in protein-free medium.

Abstract IgE-binding factor after stimulation with T cell mitogens or anti-CD3 antibodies has been shown to produce an IgE-binding protein in human T cell hybridoma AC5. The IgEBF detection by ELISA and Western blot correlated well with the previously used rosette inhibition assay. In vitro, purified IgE in the monoclonal human myeloma line U266, but not IgA or IgM-produced myelomas, providing evidence for the direct and specific regulatory action of this molecule on IgE-producing cells.

Source link: https://doi.org/10.4049/jimmunol.148.9.2834


Serologic, biologic, and western blot analysis of a T suppressor factor with specificity for the hapten 4-hydroxy-3-nitrophenyl acetyl derived from serum-free medium.

Abstract The T cell hybridoma was converted to long-term growth in serum-free medium and its product tested by serology, bioactivity, and Western blot analysis.

Source link: https://doi.org/10.4049/jimmunol.145.11.3570


Activation of human C1r: Western blot analysis reveals slow and dose-dependent activation.

Abstract A Western blot investigated C1r's spontaneous activation in the presence of EDTA. Partially purified native C1r was prepared by ultracentrifugation of fresh serum in a 10 to 30% sucrose gradient; the final concentration of C1r was one-sixth of the original serum.

Source link: https://doi.org/10.4049/jimmunol.141.5.1610


Characterization of sequential immune complexes in infective endocarditis by Western blot analysis.

Embolism related to Lactobacillus casei in a patient with cutaneous vaping was investigated. At the time of diagnosis and four weeks of therapy, immunoglobe complexes were isolated from serum. Purification of IC used differential polyethylene glycol precipitation and competitive binding to staphylococcal protein A. rabbits were injected with purified IC during the anti-IC antisera. This component responded to an antiserum that was not specific to L. casei by affinity chromatography. The restricted antigen-antibody presence in IC contrasted with a larger group of antibody circulating in patient serum. The Western blot analysis shows to be an excellent tool for characterizing IC because of its sensitivity, dissociative ability, and preservation of immunoreactivity. An IC isolated at a time far removed from the original antigenic challenge may reveal more insight into the source of the inciting antigen.

Source link: https://doi.org/10.4049/jimmunol.133.1.217


Western Blot analysis of the antigens of Toxoplasma gondii recognized by human IgM and IgG antibodies.

Both IgM and IgG antibodies were found in human blood infected with Toxoplasma gondii, according to a single major antigen with apparent m. w. In addition, IgG antibodies recognized at least 17 other antigenic substances. After subcellular fractionation, enrichment of the three key antigens identified by IgM and IgG antibodies by the membrane fraction was observed. Until IgM or IgG antibodies, Solubilization of membrane-enriched preparations with a blend of sodium dodecyl sulfate and sodium deoxycholate did not reveal any new antigenic structures that responded to IgM or IgG antibodies. Both IgM and IgG antibodies reduced the reactivity of the antigens treated with Toxoplasma lysate preparations and some fractions obtained after differential centrifugation with NaIO4 diminished the reactivity of the antigens with both IgM and IgG antibodies. IgM antibodies had no effect on the number or nature of antigens recognized by Lipase therapy. The staining of polyacrylamide gels of Toxoplasma sonicates by periodic acid-Schiff showed the presence of three components corresponding to m. w.

Source link: https://doi.org/10.4049/jimmunol.131.2.977


A Systematic Review: Comparison of Immunocytochemistry, ELISA, and Western Blot Methods in Alkaline phosphatase Measurement at Genistein-induced Osteoblast Cell

Osteoporosis, a bone disorder characterized by bone loss along with bone micro-architecture damage, is a risk of fractures. Estrogeny is one of the root of osteoporosis. Genistein is a phytoestrogen compound in the isoflavone group with a similar structure to 17u03b2-estradiol, allowing it to bind to estrogen receptors and produce an estrogenic effect. In osteoblast cells, immunocytochemistry or Enzyme-linked Immunosorbent Assay or Western blot methods can result in bone formation and promote alkaline phosphate production. ALP in osteoblast induced by genistein's selectivity, sensitivity, processing time, and cost effectiveness metrics can be determined by comparing immunocytochemistry and ELISA techniques in order to investigate ALP's osteoblast response to genistein. The western blot method has sensitivity for detecting complex-level protein expression.

Source link: https://doi.org/10.13005/bpj/2523

* Please keep in mind that all text is summarized by machine, we do not bear any responsibility, and you should always check original source before taking any actions

* Please keep in mind that all text is summarized by machine, we do not bear any responsibility, and you should always check original source before taking any actions