Flow Cytometry (FACS) Antibody Verified by Antigen Blocking

Purified recombinant antigen may competitively bind recognition site of an antibody, the antibody binding of natural antigens would be significantly decreased. This method is one of the most conventional antibody specificity verification methods. Antibody used in flow cytometry validated by antigen blocking experiments would have better specificity to intracellular/ membrane bound antigen.

For example,

Antigen: Human ICOSLG (CD275)
Antibody: ICOS Ligand / B7-H2 Antibody (FITC), Mouse MAb (11559-MM01)
Cell: RPMI8226
Flow Cyt: Use 1 µg for 10^6 cells
Instrument: BD FACSCalibur

Flow Cytometry / FACS Background

Flow cytometry is a method to evaluate cell membrane proteins and intracellular proteins as well as peptides and DNA. The principle behind FACS is an antigen-antibody reaction, with the antibodies being fluorescently labelled. There are three fluorescent proteins (R-PE, APC, and PerCP) conjugated to antibodies. Flow cytometry quantification is carried out with intercalating color labels (without the antibody). Flow cytometry antibodies are widely used in cell counting, cell sorting, biomarker detection and protein engineering.