Anti-PGP9.5 Antibody Picoband® (monoclonal, 3E4)

Western blot analysis of PGP9.5 using anti-PGP9.5 antibody (M01018-6).
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 30ug of sample under reducing conditions.
Lane 1: human U87 whole cell lysates,
Lane 2: human SH-SY5Y whole cell lysates,
Lane 3: rat brain tissue lysates,
Lane 4: mouse brain tissue lysates.
After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA for 50-90 minutes. Blocked the membrane with 5% Non-fat Milk/ TBS for 1.5 hour at RT. The membrane was incubated with mouse anti-PGP9.5 antigen affinity purified monoclonal antibody (Catalog # M01018-6) 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-mouse IgG-HRP secondary antibody at a dilution of 1:10000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1001) with Tanon 5200 system. A specific band was detected for PGP9.5 at approximately 27KD. The expected band size for PGP9.5 is at 27KD.

IHC analysis of PGP9.5 using anti-PGP9.5 antibody (M01018-6).
PGP9.5 was detected in paraffin-embedded section of human renal clear cell carcinoma tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2μg/ml mouse anti-PGP9.5 Antibody (M01018-6) overnight at 4°C. Biotinylated goat anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # SA1021) with DAB as the chromogen.

IHC analysis of PGP9.5 using anti-PGP9.5 antibody (M01018-6).
PGP9.5 was detected in paraffin-embedded section of rat brain tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2μg/ml mouse anti-PGP9.5 Antibody (M01018-6) overnight at 4°C. Biotinylated goat anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # SA1021) with DAB as the chromogen.

Flow Cytometry analysis of 293T cells using anti-PGP9.5 antibody (M01018-6).
Overlay histogram showing 293T cells stained with M01018-6 (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 mouse anti-PGP9.5 Antibody (M01018-6, 1μg/1x10⁶ cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-mouse IgG (BA1126, 5-10μg/1x10⁶ cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was mouse IgG (1μg/1x10⁶) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.