Vasantha Niranjan Chellathurai, Retnaraj Samue Selvan Johnson, Saravanakumar Venkatachalam, Jackson Durairaj Selvan
This The two authors are considered as the first author.
Regeneration and Stem Cell Biology Lab, Centre for Molecular and Nanomedical Sciences, International Research Centre,Sathyabama Institute of Science and Technology, Chennai, Tamilnadu, India.
Rep Biochem Mol Biol. 2020 Oct;9(3):264-269. doi: 10.29252/rbmb.9.3.264.
Studying protein-protein and protein-DNA interactions are prerequisites for the identification of function and mechanistic role of various proteins in the cell. Protocols for analyzing DNA-based Protein-Protein and Protein-DNA interactions are complicated and need to be simplified for efficient tracking of binding capabilities of various proteins to specific DNA molecules. Here, we demonstrated a simple yet efficient protocol for the identification of DNA coating-based Protein-DNA interaction using antibodymediated immunodetection.
Briefly, we have coated specific DNA in the microtiter plate followed by incubating with protein lysate. Specific protein-DNA and/or protein-protein bind with DNA interactions are identified using specific fluorophore-conjugated antibodies. Antibodies are used to detect a protein that is bound to the DNA.
Fluorescent-based detection identifies the specific interaction between Protein-DNA with respect to coated DNA fragments. The protocol uses indirect conjugated antibodies and hence the technique is sensitive for effective identification of Protein-DNA interactions.
Based on the results we conclude that the demonstrated protocol is simple, efficient and sensitive for identification of Protein-DNA interactions.
研究蛋白质-蛋白质和蛋白质-DNA相互作用是确定细胞中各种蛋白质功能和作用机制的前提条件。分析基于DNA的蛋白质-蛋白质和蛋白质-DNA相互作用的实验方案很复杂，需要简化，以便有效地追踪各种蛋白质与特定DNA分子的结合能力。在此，我们展示了一种简单而有效的方案，用于使用抗体介导的免疫检测来鉴定基于DNA包被的蛋白质-DNA相互作用。
简而言之，我们在微量滴定板中包被特定DNA，然后与蛋白质裂解物孵育。使用特异性荧光团偶联抗体鉴定特定的蛋白质-DNA和/或蛋白质-蛋白质与DNA的结合相互作用。抗体用于检测与DNA结合的蛋白质。
基于荧光的检测可鉴定蛋白质-DNA与包被的DNA片段之间的特异性相互作用。该方案使用间接偶联抗体，因此该技术对于有效鉴定蛋白质-DNA相互作用很敏感。
根据结果我们得出结论，所展示的方案对于鉴定蛋白质-DNA相互作用简单、高效且灵敏。
