15 μg of Jurkat lysate was 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. NSMCE1 band was visualized using ECL Western Blotting Substrate.

Result: antibody can detect NSMCE1 by Western blotting.

IHC-P analysis of testis tissue by NSMCE1 antibody.
IHC-P was performed using sections of the formalin-fixed paraffin-embedded testis 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 NSMCE1 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: Cells in seminiferous ducts are positively stained at the nuclei.

Immunoprecipitation was performed by incubation of 2.5 μg of antibody with Jurkat 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. NSMCE1 at 1 μg/mL was used as the primary antibody and peroxidase conjugated goat anti-mouse IgG (Fc specific) was used as the secondary antibody.

Lane 1: Jurkat lysate

Lane 2: NSMCE1 immunoprecipitated from Jurkat lysate by antibody

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

Result: antibody can immunoprecipitate NSMCE1.