Various protein samples were run on 6-18% SDS-PAGE under reducing conditions and blotted onto nitrocellulose membrane. antibody at 1 μg/mL was used as the primary antibody and peroxidase conjugated goat anti-mouse IgG was used as the secondary antibody. NAGK band was visualized using ECL Western Blotting Substrate.

Lane 1: 15 μg of HeLa lysate

Lane 2: 15 μg of Jurkat lysate

Lane 3: 15 μg of HEK-293 lysate
Lane 4: 15 μg of A-431 lysate

Result: antibody can detect NAGK by Western blotting.

IHC-P analysis of tonsil tissue by NAGK 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 NAGK antibody at 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.

Immunoprecipitation was performed by incubation of 2.5 μg antibody with HeLa lysate containing 200 μg total protein. After absorption with Protein G beads, the mixture was run on 6-18% SDS-PAGE and blotted onto nitrocellulose membrane. NAGK 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: HeLa lysate

Lane 2: NAGK immunoprecipitated from HeLa lysate by antibody

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

Result: antibody can immunoprecipitate NAGK.