Protein carbonylation is the most commonly used measure of oxidative modification of proteins. Quantitation of protein carbonylation by dot blot. This level of sensitivity allowed measurement of protein carbonylation in individual Drosophila. Sixty ng protein is sufficient to measure protein carbonyl content. The detection limit is 0.19 ± 0.04 pmol carbonyl. It also readily dissolves 2,4-dinitrophenylhydrazine and wets PVDF membranes. Dimethyl sulfoxide was employed as the solvent because it very efficiently extracts proteins from tissues and keeps them soluble. We developed an immunochemical dot blot method for quantitation of protein carbonylation in homogenates or purified proteins. It is frequently measured spectrophotometrically or immunochemically by derivatizing proteins with the classical carbonyl reagent, 2,4-dinitrophenylhydrazine. Quantitation of Protein Carbonylation by Dot Blot The 5-mC dot blot is a reliable, simple, and rapid method to detect the general DNA methylation level to evaluate chondrocyte phenotype. We found that the DNA methylation level differed between the monolayer subcultures, and therefore could play a key role in chondrocyte dedifferentiation. In addition, dot blots can detect overall DNA methylation level using a commercially available 5-mC antibody. Compared with other gel electrophoresis-based blotting approaches and other complex blotting procedures, the dot blot method saves significant time. In this dot blot approach, a gDNA mixture containing the methylated DNA to be detected was spotted directly onto an N + membrane as a dot inside a previously drawn circular template pattern. The gDNA methylation level was detected using a methylation-specific dot blot assay. In this study, genomic DNA (gDNA) was extracted from human chondrocytes cultured with varying number of passages. Additionally, the prohibitively high cost of HPLC instruments limits HPLC's wider application. However, HPLC requires complete digestion of genomic DNA. Other methods used to quantify global DNA methylation levels include high-performance liquid chromatography (HPLC). Due to DNA degradation during bisulfite conversion, these methods typically require a large sample volume. Current genome-wide methylation analysis techniques largely rely on bisulfite genomic sequencing. Therefore, it is important to establish a precise, simple, and rapid method to quantify global DNA methylation levels during chondrocyte dedifferentiation. However, results based on different experimental methods can be inconsistent. The global DNA methylation level of chondrocytes is considered to be a suitable biomarker for the loss of the chondrocyte phenotype. The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. Jia, Zhaofeng Liang, Yujie Ma, Bin Xu, Xiao Xiong, Jianyi Duan, Li Wang, Daping All rights reserved.Ī 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro. This article describes details of the development of an automated identity assay for a 15-valent pneumococcal conjugate vaccine, PCV15-CRM197, using capillary Western technology. An advantage this capillary Western technique has over the traditional manual dot blot Western method is the speed and the automation of electrophoresis separation, blotting, and detection steps performed in 96 capillaries. Both techniques are usually very tedious, labor-intensive, and complicated for multivalent vaccines, and they can be difficult to transfer to other laboratories. Traditionally, identity assays for biological products are performed using either an enzyme-linked immunosorbent assay (ELISA) or a manual dot blot Western. Simple Western is a new technology that allows for the separation, blotting, and detection of proteins similar to a traditional Western except in a capillary format. Hamm, Melissa Ha, Sha Rustandi, Richard R Automated capillary Western dot blot method for the identity of a 15-valent pneumococcal conjugate vaccine.
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