Anti-Human AMACR monoclonal antibody

Immunofluorescent analysis of Alpha methylacyl-CoA-racemase (green) showing staining in the in the peroxisomes and mitochondria of human prostate carcinoma tissue (right) compared to a negative control without primary antibody (left). Formalin-fixed tissue was permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Tissue was probed with an Alpha methylacyl-CoA-racemase monoclonal antibody in 3% BSA-PBS at a dilution of 1:20 and incubated overnight at 4°C in a humidified chamber. Tissue was washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. Nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 60x.

Immunohistochemistry analysis of Alpha methylacyl-CoA-racemase showing staining in the cytoplasm of paraffin-embedded human prostate carcinoma tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with an Alpha methylacyl-CoA-racemase monoclonal antibody diluted in 3% BSA-PBS at a dilution of 1:20 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Western blot analysis of Alpha methylacyl-CoA-racemase was performed by loading 25 ug of LNCAP (lane 1), HepG2 (lane 2) and mouse kidney (lane 3) cell lysates onto an SDS polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked at 4°C overnight. The membrane was probed with a Alpha methylacyl-CoA-racemase monoclonal antibody at a dilution of 1:200 (LNCAP) and 1:100 (HepG2 and mouse kidney) overnight at 4°C, washed in TBST, and probed with an HRP-conjugated secondary antibody for 1 hr at room temperature in the dark. Chemiluminescent detection was performed using Pierce ECL Plus Western Blotting Substrate. Results show a band at ~42kDa.