Anti-HRD1/SYVN1 Antibody Picoband®

Western blot analysis of HRD1/SYVN1 using anti-HRD1/SYVN1 antibody (A02670-3).
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 Hela whole cell lysates,
Lane 2: human T-47D whole cell lysates,
Lane 3: human MOLT-4 whole cell lysates,
Lane 4: rat pancreas tissue lysates,
Lane 5: mouse pamcreas 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-HRD1/SYVN1 antigen affinity purified polyclonal antibody (Catalog # A02670-3) 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 HRD1/SYVN1 at approximately 76 kDa. The expected band size for HRD1/SYVN1 is at 68 kDa.

IF analysis of HRD1/SYVN1 using anti-HRD1/SYVN1 antibody (A02670-3).
HRD1/SYVN1 was detected in an immunocytochemical section of PC-3 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-HRD1/SYVN1 Antibody (A02670-3) overnight at 4°C. DyLight550 Conjugated Goat Anti-Rabbit IgG (BA1135) 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.

Immunoprecipitating (IP) HRD1/SYVN1 in HepG2 whole cell lysate.
Western blot analysis of HRD1/SYVN1 using anti-HRD1/SYVN1 antibody (A02670-3);
Lane 1: HepG2 whole cell lysates (30ug);
Lane 2: Rabbit control IgG instead of anti-HRD1/SYVN1 antibody in HepG2 whole cell lysate;
Lane 3: anti-HRD1/SYVN1 antibody (2μg) + HepG2 whole cell lysate (500μg).
After electrophoresis, proteins were transferred to a membrane. Then the membrane was incubated with rabbit anti-HRD1/SYVN1 antigen affinity purified polyclonal antibody (A02670-3) at a dilution of 0.5 μg/mL and probed with a goat anti-rabbit IgG-HRP secondary antibody (Light Chain). The signal is developed using ECL Plus Western Blotting Substrate (Catalog # AR1196-200). A specific band was detected for HRD1/SYVN1 at approximately 76 kDa. The expected band size for HRD1/SYVN1 is at 68 kDa.

Flow Cytometry analysis of U251 cells using anti-HRD1/SYVN1 antibody (A02670-3).
Overlay histogram showing U251 cells stained with A02670-3 (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-HRD1/SYVN1 Antibody (A02670-3, 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.