Cystic fibrosis (CF) is caused by mutations in CFTR, a chloride channel involved in epithelial ion transport. Deletion of Phe508 of CFTR (DF508del) causes loss of function through both protein misfolding and reduced trafficking to the plasma membrane and impaired channel gating. Most common mutation in patients is ΔF508del; 90% have one copy and 50% have two copies.
Key Observations: Compounds that increase ΔF508del CFTR plasma membrane density (“correctors”) correlate with an increase in total CFTR protein levels. Protein analysis, such as western blotting, is the most definitive tool to observe and determine the effectiveness of compound in increasing CFTR at the plasma membrane.
Question: Can we develop a fluorescent based western blot method to detect and quantitate the levels of CFTR proteins?
Challenge: Currently, CFTR protein detection method is primarily done by chemiluminescence method and quantitation of the protein bands is semi-quantitative due to the short linear range of detection. Not only is quantitation an issue, the conventional method is very time consuming usually taking 2 days for completion.
Goal of project:
Establish fluorescent based western blot assay, including optimization of primary and secondary antibodies.
Assess the linearity of CFTR protein quantitation in the fluorescent based western blot.
Establish a western blot assay decreasing the amount of time spent for completion.