Flow Cytometric Detection of Intracellular Proteins in K562 Human Erythroleukemia Cells Using Unconjugated Primary Antibodies

Flow cytometry is a powerful technique for quantitative analysis of protein expression at single-cell resolution. This protocol describes a workflow for detecting intracellular proteins in K562 human erythroleukemia cells using unconjugated primary polyclonal antibodies and fluorophore-conjugated secondary antibodies. Cells are sequentially fixed, permeabilized, and blocked before antibody staining. By varying the order of antibody incubation relative to fixation and permeabilization, the protocol enables assessment of proteins with surface or intracellular localization using separate staining workflows. Nuclear staining of splicing factor 3a subunit 1 (SF3A1) is demonstrated using an affinity-purified rabbit anti-SF3A1 polyclonal antibody, and antibody specificity is evaluated by dose-dependent inhibition of staining with the corresponding antigenic peptide. In addition, staining conditions are used to assess reactivity of an antibody targeting the intracellular domain of erythropoietin receptor. This standardized workflow expands the use of unconjugated primary antibodies in flow cytometry and provides a practical approach for evaluating protein expression and localization, as well as antibody specificity in K562 human erythroleukemia cells.