Anti-P2RY5/LPAR6 Antibody Picoband®

Western blot analysis of LPAR6 using anti-LPAR6 antibody (A05725-1).
Electrophoresis was performed on a 10% SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions.
Lane 1: human Hacat whole cell lysates,
Lane 2: human RT4 whole cell lysates,
Lane 3: human T47D whole cell 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-LPAR6 antigen affinity purified polyclonal antibody (A05725-1) 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 (Catalog # BA1054) at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an ECL Plus Western Blotting Substrate (Catalog # AR1196-200) with Tanon 5200 system. A specific band was detected for LPAR6 at approximately 39 kDa. The expected band size for LPAR6 is at 39 kDa.

IHC analysis of LPAR6 using anti-LPAR6 antibody (A05725-1).
LPAR6 was detected in a paraffin-embedded section of human ovarian teratoma 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-LPAR6 Antibody (A05725-1) 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 LPAR6 using anti-LPAR6 antibody (A05725-1).
LPAR6 was detected in an immunocytochemical section of HepG2 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-LPAR6 Antibody (A05725-1) overnight at 4°C. Fluoro488 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.

Flow Cytometry analysis of PC-3 cells using anti-LPAR6 antibody (A05725-1).
Overlay histogram showing PC-3 cells stained with A05725-1 (Blue line). The cells were fixed with 4% paraformaldehyde and blocked with 10% normal goat serum. And then incubated with rabbit anti-LPAR6 Antibody (A05725-1, 1 μg/1x10⁶ cells) for 30 min at 20°C. Fluoro488 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.