If I am studying the gene which is tagged by fluorescent tag endogenously by CRISPR/Cas9 Knock-in or if the same gene over-expressed and tagged with same fluorescent tag. Then what difference it will make in the study and observation by Confocal microscopy on the functional study of the protein.
Basically it depends on the gene and what you want to study about it. However, generally overexpression can potentially cause some abnormalities in protein function, localization and also cellular physiology, based on the protein that you want to study. Therefore, theoretically tagging the endogenous gene is supposed to be the more accurate strategy. Although, overexpression strategy may still work for many genes.
It is a good idea to study published data on overexpression of the protein to see if there is any data suggesting that the overexpression does not significantly change the aspects of protein function that you want to study.
Generally to some extent I agree with Mohsen, but prior to any knock-in or over-expression study you need to do some biochemical assay on your protein of interest because it has been shown that most of the time tags make proteins partially unfolded especially those with enzymatic activities ( e.g., HDACs or Kinases). If you show that your protein is biochemically active and properly folded you can continue your study otherwise it is wasting time and money. For example, as a first step you better to check your protein folding by NMR etc. Say it is a DNA binding protein you better check its binding affinity or if it is an enzyme first check its activity on its substrate using tagged and untagged protein.
Thank you so much @Mehdi Sharifi Tabar and @Mohseen Basiri, for your valuable comments I got my answer now. thanks again.
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