Hi everyone,
There are challenges with using antibodies to capture my protein of interest so I will be using CRISPR to endogenously tag my RNA binding protein with a fluorophore followed by a His tag (using linker sequences) for protein purification for mass spec in iPSCs. I am aware any tag onto your endogenous protein will have an effect, however will using His tag pose a greater problem than using a FLAG or HA tag due to its positive charge? I can't seem to find any papers whereby they have stably tagged an endogenous protein with His tag. Any comments/feedback would be appreciated.
@all Using a His tag for protein purification is a commonly used approach, and there are several reports of successful tagging of endogenous proteins with a His tag. One potential challenge with using a His tag is that it may interact non-specifically with other proteins or cellular components due to its positive charge, which could affect the function of the tagged protein or lead to false positives in mass spectrometry analysis. However, this can be minimized by optimizing the purification conditions and using appropriate controls.
In general, the choice of tag depends on several factors such as the protein of interest, the downstream applications, and the availability of antibodies or other reagents. FLAG and HA tags are also commonly used for protein tagging and purification, and they have their own advantages and disadvantages. FLAG tags are smaller in size and can be more accessible for antibody detection, but they may be more prone to degradation. HA tags are larger and may be more difficult to detect with antibodies, but they are less likely to interfere with protein function.
Ultimately, the choice of tag depends on the specific needs of the experiment, and it may be worth considering testing multiple tags to determine which one works best for your protein of interest.
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