IHC-P analysis of tonsil tissue by IRAG2 antibody.
IHC-P was performed using sections of the formalin-fixed paraffin-embedded tonsil tissue. Antigen was retrieved through addition of boiling Tris/EDTA buffer pH 9 in a pressure cooker for 3 min. Endogenous peroxidase activity was quenched by incubating the sections with 3% H₂O₂ for 30 min at room temperature. The sections were then incubated with IRAG2 antibody at 7.5 μg/mL at room temperature for 1 h. Poly-peroxidase conjugated goat anti-mouse IgG was used as the secondary antibody. Diaminobenzidine was used as the chromogen. The section was counterstained with hematoxylin. A tissue section incubated with phosphate-buffered saline followed by incubation with the secondary antibody was used as the background control.

Result: Germinal center cells and non-germinal center cells are positively stained at the cytoplasm and membrane.

Immunoprecipitation was performed by incubation of 2.5 μg of antibody with Ramos lysate containing 200 μg of total protein. After absorption with Protein G beads, the mixture was run on 6-18% SDS-PAGE and blotted onto nitrocellulose membrane. IRAG2 at 1 μg/mL was used as the primary antibody and peroxidase conjugated rabbit anti-mouse IgG (Light chain specific) was used as the secondary antibody.

Lane 1: Ramos lysate

Lane 2: IRAG2 immunoprecipitated from Ramos lysate by antibody

Lane 3: The same as Lane 2 but KT82 was used as IgG isotype control antibody

Result: antibody can immunoprecipitate IRAG2.