Here we describe the method for fast detection of total m6A levels in mRNA by dot blot analysis using a specific m6A antibody. with N1-Methyladenosine (m1A) (E8S7H) Rabbit mAb (upper) or N6-Methyladenosine (m6A) (D9D9W) Rabbit mAb 56593 (lower). Hapten N6-methyladenosine-5-mono-phosphate conjugated to BSA of all N6-methyladenosine (m6A). The total amount of m6A can be detected by several methods, such as dot blot analysis using specific m6A antibodies and quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) (Fu et al., 2014 Shen et al., 2016). Bring this labeled antibody directly to your bench. N6-methyladenosine (m6A) is the most prevalent internal modification of eukaryotic messenger RNA (mRNA). Dot Blot Analysis of N6-methyladenosine RNA Modification Levels. The most important factor determining the success of the modified northern blotting protocol is the integrity of the RNA sample. Dot blot protocol technique for detecting, analyzing and identifying proteins, similar to the western blot technique but differing in that protein samples are not separated electrophoretically but are spotted through circular templates directly onto the membrane or paper substrate. Dot Blot Analysis of N6-methyladenosine RNA Modification Levels In the future, it could also be possible to combine the experimental data from both the classical northern blotting and the modified northern blotting protocols, providing greater insights into RNA biology. Abstract Posttranscriptional modification of mRNAs plays an important role in establishing the functional diversity of the proteome.
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