Low Recovery Due to Incompatible Antibody Affinity Beads (ChIP (Low Signal))

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Low signal across all samples with high background. Antibody appears present in supernatant after IP, suggesting poor capture by beads. Common causes: 1) Protein A or Protein G alone has low affinity for antibody isotype being used 2) Mismatch between bacterial protein (A or G) and immunoglobulin class 3) Certain IgG subclasses bind preferentially to either Protein A or Protein G 4) Using only one type of affinity bead reduces capture efficiency Solutions: 1) Use a mixture of Protein A and Protein G coupled to sepharose beads 2) Typical ratio: 50:50 mix of Protein A/G-sepharose for broad antibody compatibility 3) For rabbit antibodies: Protein A generally works well 4) For mouse IgG1: Protein G shows better binding; for mouse IgG2a/2b: use Protein A 5) Consider using pre-coupled Protein A/G agarose beads (commercially available)