Using GFP Fusion Proteins to Study Protein Localization
Author Information
Author(s): Ella Palmer, Tom Freeman
Primary Institution: MRC Rosalind Franklin Centre for Genomics Research
Hypothesis
Does the position of GFP tagging (N-terminal vs C-terminal) affect the subcellular localization of proteins?
Conclusion
C-terminal tagging with GFP generally preserves the native localization of proteins better than N-terminal tagging.
Supporting Evidence
- All C-terminal fusion proteins localized correctly, while less than half of N-terminal fusion proteins did.
- Nine C-terminal-tagged proteins showed transfection in 80% or more spots.
- Five N-terminal fusion proteins showed no transfection events at all.
Takeaway
This study looked at how attaching a green fluorescent protein (GFP) to the start or end of other proteins affects where those proteins end up in a cell. It found that tagging at the end usually works better.
Methodology
The study used reverse transfection microarrays to analyze the localization of 16 proteins tagged with GFP at either the N- or C-terminal.
Limitations
The study is limited by the small sample size and the potential for errors in gene cloning.
Digital Object Identifier (DOI)
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