Using chemiluminescence imaging of cells (CLIC) for relative protein quantification

Area of Science:

• Cellular biology
• Biochemistry
• Immunology

Background:

• Protein quantification is crucial for understanding cellular processes and disease.
• Existing methods like immunofluorescence can be limited by auto-fluorescence.
• Accurate protein analysis is essential for cell physiology and pathophysiology research.

Purpose of the Study:

• To introduce a simple, sensitive, and robust chemiluminescence-based immunoassay (CLIC) for relative protein quantification in whole cells.
• To demonstrate the utility of CLIC for quantifying various cellular events, including complement activation and protein dynamics.
• To validate CLIC against established quantification methods and highlight its advantages.

Main Methods:

• Development of a chemiluminescence imaging of cells (CLIC) assay.
• Application of CLIC for quantifying complement activation, membrane protein expression, shedding, binding, and internalization.
• Utilizing specific membrane permeabilization for cytosolic and nuclear protein quantification and translocation analysis.
• Validation of CLIC through parallel experiments with ELISA, qPCR, and immunofluorescence microscopy.

Main Results:

• CLIC provides sensitive and robust relative quantification of proteins in whole cells.
• The method successfully quantified complement activation and various membrane protein dynamics.
• CLIC enabled the analysis of cytosolic, nuclear proteins, and their translocation.
• Validation confirmed CLIC's performance comparable to ELISA, qPCR, and immunofluorescence microscopy.

Conclusions:

• CLIC is a versatile and advantageous immunoassay for relative protein quantification.
• The assay is suitable for both small-scale and high-throughput studies due to its simplicity and common reagent use.
• CLIC offers good linearity, dynamic range, and signal stability, making it a valuable tool in cellular research.