Anti-AMACR Antibody Picoband®

Western blot analysis of AMACR using anti-AMACR antibody (PB9983).
Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions.
Lane 1: human 293T whole cell lysates,
Lane 2: human LNCAP whole cell lysates,
Lane 3: human HepG2 whole cell lysates,
Lane 4: human Hela whole cell lysates,
Lane 5: rat kidney tissue lysates,
Lane 6: rat liver tissue lysates,
Lane 7: mouse kidney tissue lysates,
Lane 8: mouse liver tissue lysates.
After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-AMACR antigen affinity purified polyclonal antibody (Catalog # PB9983) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for AMACR at approximately 42 kDa. The expected band size for AMACR is at 42 kDa.

IHC analysis of AMACR using anti-AMACR antibody (PB9983).
AMACR was detected in a paraffin-embedded section of human stomach cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml rabbit anti-AMACR Antibody (PB9983) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

IHC analysis of AMACR using anti-AMACR antibody (PB9983).
AMACR was detected in a paraffin-embedded section of mouse kidney tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml rabbit anti-AMACR Antibody (PB9983) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

IHC analysis of AMACR using anti-AMACR antibody (PB9983).
AMACR was detected in a paraffin-embedded section of rat kidney tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml rabbit anti-AMACR Antibody (PB9983) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

IF analysis of AMACR using anti-AMACR antibody (PB9983).
AMACR was detected in an immunocytochemical section of U2OS cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (AR0022) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 5 μg/mL rabbit anti-AMACR Antibody (PB9983) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Rabbit IgG (BA1127) was used as secondary antibody at 1:500 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

IF analysis of AMACR and TP63 using anti-AMACR antibody (PB9983) and anti-TP63 antibody (PB9152).
AMACR and TP63 were detected in paraffin-embedded sections of human prostatic hyperplasia tissue. Heat-mediated antigen retrieval was performed in EDTA buffer (pH 8.0). Tissue sections were blocked with 5% BSA. The sections were incubated overnight with 5 μg/mL rabbit Anti-TP63 antibody (PB9152). HRP-labeled secondary antibody was added dropwise and incubated for 30 minutes. TYR-570plus fluorescent dye reaction solution was then added dropwise and incubated at room temperature in the dark. Next, the sections were immersed in antigen retrieval buffer again to elute the bound antibodies. The staining steps were then repeated for the second round of labeling. Anti-AMACR antibody (PB9983) was diluted 1:50 and incubated with the sections overnight at 4°C. HRP-labeled secondary antibody was added dropwise and incubated for 30 minutes. After washing, TYR-520 plus fluorescent dye reaction solution was added dropwise and incubated at room temperature in the dark. Finally, DAPI staining solution was added dropwise to stain the nuclei, and the slides were sealed with an anti-fade mounting medium. The results were observed under a fluorescence microscope.

Flow Cytometry analysis of HepG2 cells using anti-AMACR antibody (PB9983).
Overlay histogram showing HepG2 cells stained with PB9983 (Blue line). To facilitate intracellular staining, cells were fixed with 4% paraformaldehyde and permeabilized with permeabilization buffer. The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-AMACR Antibody (PB9983, 1 μg/1x10⁶ cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-rabbit IgG (BA1127, 5-10 μg/1x10⁶ cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was rabbit IgG (1 μg/1x10⁶) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.