PMEL Antibody for IF / Premelanosome Protein Antibody (FY13157)

PMEL Antibody for IF / Premelanosome Protein Antibody immunofluorescence analysis in human melanoma tissue. FFPE human melanoma section shows strong red fluorescent cytoplasmic staining in melanoma cells corresponding to premelanosome-associated localization of PMEL (SILV/gp100). The staining pattern appears as punctate vesicular fluorescence within the cytoplasm of tumor cells, consistent with melanosome and premelanosome structures characteristic of melanocytic lineage cells. Nuclei are counterstained with DAPI (blue). Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0) prior to antibody incubation, followed by detection with DyLight 594-conjugated goat anti-rabbit IgG secondary antibody and visualization by fluorescence microscopy.

Western blot analysis of SILV/PMEL using anti-PMEL antibody. Lane 1: human whole cell lysates, Lane 2: rat testis tissue lysates, Lane 3: rat C6 whole cell lysates, Lane 4: mouse testis 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-PMEL antibody at 0.5 ug/ml overnight at 4oC, 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 was developed using enhanced chemiluminescent. PMEL antibody detects a band at ~75-80 kDa in the indicated lysates. Although the full-length glycosylated PMEL precursor is ~100 kDa, it is rapidly cleaved into luminal M-alpha (~60-80 kDa) and membrane M-beta (~26 kDa) fragments. The ~75-80 kDa species corresponds to the glycosylated M-alpha fragment, the predominant soluble intermediate form of PMEL detected in standard cell and tissue lysates.

PMEL Antibody immunohistochemistry analysis in human melanoma tissue. FFPE human melanoma section shows HRP-DAB brown cytoplasmic and membranous staining in melanoma tumor cells, consistent with premelanosome-associated localization of PMEL (SILV/gp100) in melanocytic lineage cells. Staining highlights sheets of melanoma cells while surrounding stromal regions show minimal background signal. Heat-mediated antigen retrieval was performed in EDTA buffer (pH 8.0) prior to antibody incubation, followed by detection using peroxidase-conjugated goat anti-rabbit IgG secondary antibody and visualization with HRP-DAB chromogenic substrate.

Flow Cytometry analysis of U20S cells using PMEL/Premelanosome Protein antibody. Overlay histogram showing U20S cells stained with (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-PMEL antibody (1 ug/million cells) for 30 min at 20oC. DyLight 488 conjugated goat anti-rabbit IgG (5-10 ug/million cells) was used as secondary antibody for 30 minutes at 20oC. Isotype control antibody (Green line) was rabbit IgG (1 ug/million cells) used under the same conditions. Unlabelled sample (Red line) was also used as a control.

Immunohistochemical staining of SILV/PMEL using anti-PMEL antibody. SILV/PMEL was detected in a paraffin-embedded section of human melanoma 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 ug/ml rabbit anti-PMEL antibody overnight at 4oC. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37oC. The tissue section was developed using an HRP secondary and DAB substrate.