Antibody Works in Other Applications but Not Flow (Flow Cytometry (CST Guide))

Question

Accepted Answer

Antibody validated for Western blot or immunofluorescence shows no signal or high background when used in flow cytometry protocol. Common causes: 1) Antibody not recommended or validated for flow cytometry on product data sheet (either not tested or failed CST flow validation criteria) 2) Antibody concentration optimized for other applications (e.g., 1:1000 for Western) inappropriate for flow cytometry with 10⁵-10⁶ cells 3) Epitope accessibility differs between fixed/permeabilized flow samples versus native Western or IF conditions Solutions: 1) Verify antibody is validated for flow cytometry on CST product data sheet before use; contact technical support for testing status if unclear 2) For immunofluorescence-approved antibodies, perform titration series (e.g., 1:50, 1:100, 1:200) to determine optimal concentration for flow 3) When using low cell numbers (< 10⁵), perform independent titration as CST recommendations are optimized for 10⁵-10⁶ cells