{"doi":"10.1101/2023.09.04.556277","title":"DropBlot: single-cell western blotting of chemically fixed cancer cells","abstract":"To further realize proteomics of archived tissues for translational research, we introduce a hybrid microfluidic platform for high-specificity, high-sensitivity protein detection from individual chemically fixed cells. To streamline processing-to-analysis workflows and minimize signal loss, DropBlot serially integrates sample preparation using droplet-based antigen retrieval from single fixed cells with unified analysis-on-a-chip comprising microwell-based antigen extraction followed by chip-based single-cell western blotting. A water-in-oil droplet formulation proves robust to the harsh chemical (SDS, 6M urea) and thermal conditions (98°C, 1-2 hr.) required for sufficient antigen retrieval, and the electromechanical conditions required for electrotransfer of retrieved antigen from microwell-encapsulated droplets to single-cell electrophoresis. Protein-target retrieval was demonstrated for unfixed, paraformaldehyde-(PFA), and methanol-fixed cells. We observed higher protein electrophoresis separation resolution from PFA-fixed cells with sufficient immunoreactivity confirmed for key targets (HER2, GAPDH, EpCAM, Vimentin) from both fixation chemistries. Multiple forms of EpCAM and Vimentin were detected, a hallmark strength of western-blot analysis. DropBlot of PFA-fixed human-derived breast tumor specimens (n = 5) showed antigen retrieval from cells archived frozen for 6 yrs. DropBlot could provide a precision integrated workflow for single-cell resolution protein-biomarker mining of precious biospecimen repositories.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2023,"id":395047,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":3,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9518,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2023-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":420688,"name":"Amy E. Herr","orcid":"0000-0002-6906-2985","position":1,"is_corresponding":false},{"id":1013201,"name":"Yang Liu","orcid":"0000-0002-8747-4975","position":0,"is_corresponding":true}],"reference_count":68,"raw_metadata":null,"created_at":"2026-07-19T01:19:18.686170Z","pmid":"37732260","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}