Non-specific Protein Binding to Antibody (Immunoprecipitation (High Background))

Question

Accepted Answer

Multiple protein bands appear in IP eluate that are not related to the target antigen, indicating proteins binding non-specifically to the antibody Fc region or other antibody domains. Common causes: 1) Lysate contains high abundance proteins with affinity for antibody Fc regions 2) Sample overloaded with too much total protein relative to antibody binding capacity 3) Lysate not pre-cleared to remove proteins that bind non-specifically to immunoglobulins 4) Proteins with intrinsic antibody-binding properties (e.g., Protein A/G-binding proteins) present Solutions: 1) Reduce amount of sample lysate loaded onto beads if many non-specific proteins appear 2) Pre-clear lysate by pre-incubating with beads alone (without antibody) for 30-60 min at 4°C 3) Use an irrelevant antibody of same species and IgG subclass to pre-clear lysate before IP 4) Keep lysate protein concentration in range of 10-500 µg for standard IP reactions