{Reference Type}: Journal Article
{Title}: Profiling of BCLxL Protein Complexes in Non-Small Cell Lung Cancer Cells via Multiplexed Single-Molecule Pull-Down and Co-Immunoprecipitation.
{Author}: Kim SH;Chun C;Yoon TY;
{Journal}: Anal Chem
{Volume}: 96
{Issue}: 22
{Year}: 2024 06 4
{Factor}: 8.008
{DOI}: 10.1021/acs.analchem.3c05801
{Abstract}: We introduce multiplexed single-molecule pull-down and co-immunoprecipitation, named m-SMPC, an analysis tool for profiling multiple protein complexes within a single reaction chamber using single-molecule fluorescence imaging. We employed site-selective conjugation of biotin and fluorescent dye directly onto the monoclonal antibodies, which completed an independent sandwich immunoassay without the issue of host cross-reactivity. We applied this technique to profile endogenous B-cell lymphoma extra-large (BCLxL) complexes in non-small cell lung cancer (NSCLC) cells. Up to three distinct BCLxL complexes were successfully detected simultaneously within a single reaction chamber without fluorescence signal crosstalk. Notably, the NSCLC cell line EBC-1 exhibited high BCLxL-BAX and BCLxL-BAK levels, which closely paralleled a strong response to the BCLxL inhibitor A-1331852. This streamlined method offers the potential for quantitative biomarkers derived from protein complex profiling, paving the way for their application in protein complex-targeted therapies.