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Western Blot - Crossref

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Last Updated: 09 August 2022

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Runt-Related Transcription Factor 2 (Runx2) Measurement in Phytoestrogen-Induced Bone: A Comparison of Western Blot and Immunohistochemistry Methods

Estrogen deficiency can cause osteoporosis in postmenopausal women. Phytoestrogens are now widely accepted as a potential estrogen replacement therapy. The administration of phytoestrogens can cause bone synthesis, as shown by an increase in Runx2 expression in osteoblast cells, and can be seen using western blot and immunohistochemistry techniques. Using western blots and immunohistochemistry, this research sought to compare Runx2's detection methods in phytoestrogen-induced bone tissue culture. According to the results, the selectivity and sensitivity of western blot for detecting Runx2 on tissue were 93% and 99. 5%, respectively, although immunohistochemistry selectivity and sensitivity were 45-45%. Western blot can save up to 57. 2 percent compared to immunohistochemistry, compared to immunohistochemistry. Immunohistochemistry takes 46 hours to digest, while Western blot takes 25 hours and 20 minutes. The western blot is more specific, accurate, fast, and inexpensive for detecting Runx2 in bone tissue in comparison to immunohistochemistry.

Source link: https://doi.org/10.13005/bpj/2439


Method for detecting acetylated PD-L1 in cell lysates by immunoprecipitation and western blot analysis

This makes it difficult to study endogenous protein acetylation. This paper describes a robust laboratory protocol for the enrichment and detection of endogenous acetylated PD-L1 protein. From a variety of cell lines and covering a fourteen-fold range of target protein concentrations, a newly developed acetyl lysine affinity matrix was used to enrich > 90% of acetylated PD-L1 species. The endogenous PD-L1 measurement, which used a highly specific PD-L1 antibody and optimized transfer times, was used to determine that the endogenous level of acetylated PD-L1 is within the range of 0. 02–u20130. 07% of total PD-L1. As validation, we show that acetylation levels have increased to 0. 11 percent of total PD-L1 after a 4h course with the histone deacetylase inhibitor trichostatin A. In any laboratory equipped with tissue culture or western blot machines, the procedure described here is straightforward to perform.

Source link: https://doi.org/10.1371/journal.pone.0268887


Abstract 4958: Analysis of the AKT signaling pathway in prostate cancer using a streamlined Western blot workflow

Abstract Relative protein levels in the Akt pathway were determined in metastasized and normal prostate cancer cell lines using a new streamlined Western blot process and compared to a traditional Western blot approach. Protein expression levels in the Akt pathway are often determined using Western blotting, but new Western blotting procedures involve multiple time-consuming steps that can take more than 5 hours or even days. A streamlined Western blot workflow that includes a rapid 10-minute protein exchange as well as a one-hour immunoblotting procedure were used. The protocol is only 30 minutes when using an overnight primary antibody incubation. These results show that recent Western blot methods involving multiple time-consuming steps can be streamlined to produce comparable results for phospho-specific and total protein targets. Both low- and high-abundance target proteins were found in the characterization of the Akt pathway, which yielded similar sensitivity to traditional Western blotting techniques for both low- and high-abundance target proteins.

Source link: https://doi.org/10.1158/1538-7445.am10-4958


Abstract LB-288: Optimized xylene-free protein extraction method from formalin-fixed paraffin-embedded tissue sections for western blot analysis

Background: Formalin fixed paraffin embedded tissues is the most common method for fixation and storage of tissues for use in clinical pathology experiments. Proteins can be obtained with a novel process that uses hot distilled water as a substitute for xylene with a quality suitable for western blot analysis, as shown by We previously. To solve these problems, we created a new xylene-free protein extraction from FFPE tissue sections of approximately 8 u03 billion cm thickness. Methods: A total of 44 different FFPE tissues sections of 8 u03bcm thickness were obtained from various archived FFPE specimens including 17 colorectal cancer, 7 breast cancer, 3 thyroid cancer, 3 thyroid cancer, 4 ovarian cancer, 8 uterine cancer, and 5 prostate cancer. The tissues were then placed in a cell lysis buffer and Laemli buffer and incubated at 100°u00b0C for 5 minutes. The amount of proteins extracted in this experiment is at least four times greater than the standard protein extraction process using xylene. By the western blot experiment, protein extracts were of high quality and quickly analysed. In addition, the isolated proteins showed a similar migration pattern to the positive control protein on SDS-PAGE, but with increased bandsu2019 intensity and clarity, according to the researchers. Conclusion: We developed an effective, safe, cost-effective, and fast way to isolate proteins from FFPE tissue sections, which was suitable for western blot analysis. It's unclear if this method of protein extraction from FFPE is suitable for use in proteomic analyses.

Source link: https://doi.org/10.1158/1538-7445.am2015-lb-288


Abstract 2218: Qualitative and quantitative analysis of IDH1 mutation in progressive gliomas by allele-specific quantitative RT-PCR and Western blot analysis

We wanted to test this new strategy in a larger patient cohort, compare it to control tissue, and see if the expression of both, IDH1mut and IDH1 wildtype correlates with disease course and different treatment regimens. Primary glioma patients with diagnosis of diffuse glioma, anaplastic glioma, secondary glioma, secondary glioma, secondary glioma, primary glioma +/- chemotherapy, and primary glioblastoma +/- CTx were obtained intraoperatively from 84 glioma patients with diagnosis of diffuse glioma, anaplastic glioma IDH1 mRNA expression was determined by Western Blot analysis and IHC using real-time PCR with specific primers for IDH1mut and -wt; protein expression was confirmed by Western Blot analysis and IHC. Quantitative IDH1mut testing has been performed in larger patient populations and seems to be a quick and u223c10 times more accurate method for quantitative IDH1mut determination as compared to the ones used so far. Our findings with this new technology confirmed previous findings that the majority of astrocytomas and secondary GBM have the mutation, although most primary GBM do not. IDH1mut expression is upregulated in sGBM relative to lower grade glioma, according to quantitative testing. In addition, semiquantitative protein expression analysis revealed elevated expression levels of mutated IDH1 in sGBM. In addition, we observed an increase in IDH1mut gene expression in sec. Compared to lower grade glioma, IDH1mut has a pivotal role in glioma formation but also in glioma progression. By allele-specific quantitative RT-PCR and Western blot analysis, qualitative and quantitative review of the IDH1 mutation in progressive gliomas can be derived from a qualitative and quantitative investigation of IDH1 mutation in progressive gliomas.

Source link: https://doi.org/10.1158/1538-7445.am2015-2218


Abstract 5645: Critical variables to reduce Western blot variability

BlotCycleru2122, a self-conserving western blot processor, can be eliminated from the variability inherent with immunodetection and helps to achieve higher sensitivity by optimizing washing methods. During immunodetection, we used BlotCycler to identify the root of error during immunodetection. Relatively small changes in duration and temperature of incubation may have a significant influence on not only the signal volume but also the specificity of antibody-specific interaction with antigen. Optimized and control of key variables contributing to improved reproducibility and specificity of immunodetection, reliable protein quantification, primary antibody specificity determination, and optimization of primary and secondary antibody concentrations can be routinely used for new biomarker discovery and analysis. [abstract]: Western blot variability can be reduced by critical variables [abstract]. In:: The American Association for Cancer Research Annual Meeting 2018 (April 14-18) in Chicago, Illinois, is published in the United States' Proceedings; 2018 Apr 14-18; Abstracts of the 2018 Annual Meeting of the American Association for Cancer Research Annual Meeting, 2018; 2018-04-23; Chicago, Illinois.

Source link: https://doi.org/10.1158/1538-7445.am2018-5645


Abstract 224: Critical variables for automated protein immunodetection using western blot

Automated western blot processor BlotCycleru2122, a robotic western blot processor, uses a fluidic control system that helps to minimize the variability associated with immunodetection and higher sensitivity by optimizing washing method. We used BlotCycler to measure critical variables for immunodetection including temperature, incubation time, and reagent mixing. Relatively small changes in length and temperature in incubation can have a significant effect on not only the amount of evidence but also the specificity of antibody-inhibition relations with antigen. Automated processing with BlotCycler helps improve reproducibility and specificity of immunodetection, improved reproducibility and detection of immunodetection, reliable protein quantification, primary antibody specificity determination, and optimization of primary and secondary antibody concentrations. Western blot [abstract] is one of the crucial parameters for automated protein immunodetection using western blot [abstract]. In:: The American Association for Cancer Research Annual Meeting 2017 (2017-04-01) Washington, DC.

Source link: https://doi.org/10.1158/1538-7445.am2017-224


Detection of Cyclin B1 Expression in G1-Phase Cancer Cell Lines and Cancer Tissues by Postsorting Western Blot Analysis

As determined by flow cytometry, we found cyclin B1 in the suspended G1-phase MOLT-4 lymphocyte leukemia cells and in G1 phase T-7 transitional tumor cells in the present research. In addition, we reported that cyclin B1 was present in breast cancer cells culture in patient tissues and lymphocytes from patients with leukemia in the G1 phase. Cyin B1 was discovered in early G1 cells, but in MOLT-4 cells detained in G1-S phase, cyclin B1 was mostly present in late G1 phase. We find that the cyclin B1 expressed in the G1 phase may differ from that expressed in the G2-M phase, and that this unscheduled form of cyclin B1 may play a significant role in tumor formation and apoptosis.

Source link: https://doi.org/10.1158/0008-5472.can-03-3321

* 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