One-step split GFP staining for sensitive protein detection and localization in mammalian cells

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• Epitope tags are crucial for detecting intracellular proteins and their localization using antibodies.
• However, traditional antibody-based methods often suffer from problematic background and nonspecific staining, reducing signal clarity.

Purpose of the Study:

• To develop a novel, highly specific, and low-background assay for detecting intracellular proteins and their localization.
• To leverage the split green fluorescent protein (GFP) complementation system for enhanced protein detection.

Main Methods:

• A split GFP complementation system was employed, where proteins are tagged with the GFP 11 fragment.
• Detection involves adding a recombinant GFP 1-10 fragment to reconstitute a fluorescent GFP.
• The assay was validated using flow cytometry and microscopy for proteins with diverse subcellular localizations.

Main Results:

• The split GFP complementation assay demonstrated high specificity and significantly reduced background fluorescence compared to antibody-based methods.
• This resulted in substantially higher signal-to-noise ratios for protein detection.
• The assay successfully facilitated the detection of proteins across various subcellular compartments.

Conclusions:

• The split GFP tagging system provides a robust and sensitive method for detecting intracellular proteins.
• This one-step assay offers a superior alternative to traditional methods, enhancing the study of protein localization.
• The technique is applicable to both flow cytometry and microscopy, broadening its utility in biological research.