IHC-P analysis of skeletal muscle tissue by PFKM antibody.
IHC-P was performed using sections of the formalin-fixed paraffin-embedded skeletal muscle 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% H₂O₂for 30 min at room temperature. The sections were then incubated with PFKM 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: Myocytes are positively stained at the cytoplasm.

Immunoprecipitation was performed by incubation of 2.5 μg of antibody with SH-SY5Y 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. PFKM (OC-050) at 1 μg/mL was used as the primary antibody and peroxidase conjugated goat anti-mouse IgG was used as the secondary antibody.

Lane 1: SH-SY5Y lysate

Lane 2: PFKM immunoprecipitated from SH-SY5Y lysate by antibody

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

Result: antibody can immunoprecipitate PFKM.