A Ni-NTA-based red fluorescence probe for protein labelling in live cells

Area of Science:

• Biochemistry
• Molecular Biology
• Cellular Imaging

Background:

• The His6-Ni-nitrilotriaceate (Ni-NTA) system is widely used for protein purification.
• Developing fluorescent probes for live-cell imaging of intracellular proteins remains challenging due to cell membrane permeability issues.

Purpose of the Study:

• To design and synthesize a novel red fluorescent probe (Ni-NTA-AB) capable of selectively labeling His6-tagged proteins within live cells.
• To evaluate the probe's ability to enhance fluorescence upon binding and to quantify proteins.

Main Methods:

• Synthesis of a new red fluorescent probe (Ni-NTA-AB) incorporating a Ni-NTA moiety, BODIPY, and an arylazide.
• Demonstration of selective binding to His6-tagged proteins, exemplified by xerodermapigmentosum group A (XPA122).
• Assessment of intracellular labeling in live bacterial cells with Tween 80 and quantification in SDS-PAGE via photoactivation.

Main Results:

• The Ni-NTA-AB probe selectively binds His6-tagged proteins, resulting in approximately 26-fold fluorescence enhancement.
• The probe successfully enters live bacterial cells to label intracellular His6-tagged proteins.
• Quantification of His6-tagged proteins in SDS-PAGE is achievable with a wide linear range (25-1000 ng) after photoactivation.

Conclusions:

• The developed Ni-NTA-AB probe offers a promising tool for selective labeling and imaging of intracellular His6-tagged proteins.
• This probe facilitates multiple-channel tracking and labeling of His6-tagged proteins in various biological contexts.
• The study opens new avenues for visualizing and quantifying tagged proteins within live cells and in biochemical assays.