Total protein is an effective loading control for cerebrospinal fluid western blots

Area of Science:

• Neuroscience
• Biochemistry
• Proteomics

Background:

• Cerebrospinal fluid (CSF) is crucial for identifying neurological disease biomarkers.
• High-throughput methods for CSF protein biomarker discovery require robust validation.
• Accurate Western blotting (WB) of CSF is hindered by the lack of a validated loading control.

Purpose of the Study:

• To investigate total protein (TP) staining as a reliable loading control for CSF WB.
• To assess the accuracy and sensitivity of CSF WB using TP staining for biomarker validation.

Main Methods:

• Investigated TP staining using iodine-based reversible membrane staining for linearity and consistency.
• Utilized spiked green fluorescent protein (GFP) in CSF to quantify WB accuracy before and after TP correction.
• Compared WB and ELISA measurements of complement C3 and cystatin C in neurological disease and control CSF samples.

Main Results:

• CSF WB with TP loading control accurately detected GFP level differences and corrected simulated loading errors.
• WB and ELISA measurements of complement C3 and cystatin C showed significant correlation.
• TP loading correction improved the sensitivity of CSF WB for detecting between-group differences.

Conclusions:

• CSF TP staining offers superior linear dynamic range and consistency over albumin.
• The TP method effectively corrects loading errors and enhances CSF WB sensitivity.
• TP staining serves as a validated loading control, improving accuracy in CSF WB analyses.