Anti-Grp75/HSPA9 Antibody Picoband®

Western blot analysis of Grp75 using anti-Grp75 antibody (PB9642).
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 A549 whole cell lysates,
Lane 3: human Jurkat whole cell lysates,
Lane 4: human HepG2 whole cell lysates,
Lane 5: rat testis tissue lysates,
Lane 6: rat PC-12 whole cell lysates,
Lane 7: mouse testis tissue lysates,
Lane 8: mouse NIH/3T3 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-Grp75 antigen affinity purified polyclonal antibody (Catalog # PB9642) 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 Grp75 at approximately 74 kDa. The expected band size for Grp75 is at 74 kDa.

IHC analysis of Grp75 using anti-Grp75 antibody (PB9642).
Grp75 was detected in a paraffin-embedded section of human lung 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-Grp75 Antibody (PB9642) 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.

Immunoprecipitating (IP) Grp75 in A549 whole cell lysate.
Western blot analysis of Grp75 using anti-Grp75 antibody (PB9642);
Lane 1: A549 whole cell lysates (30ug);
Lane 2: Rabbit control IgG instead of anti-Grp75 antibody in A549 whole cell lysate;
Lane 3: anti-Grp75 antibody (2μg) + A549 whole cell lysate (500μg).
After electrophoresis, proteins were transferred to a membrane. Then the membrane was incubated with rabbit anti-Grp75 antigen affinity purified polyclonal antibody (PB9642) at a dilution of 0.5 μg/mL and probed with a goat anti-rabbit IgG-HRP secondary antibody (Catalog # BA1054). The signal is developed using ECL Plus Western Blotting Substrate (Catalog # AR1196-200). A specific band was detected for Grp75 at approximately 74 kDa. The expected band size for Grp75 is at 74 kDa.

IHC analysis of Grp75 using anti-Grp75 antibody (PB9642).
Grp75 was detected in a paraffin-embedded section of human intestinal 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-Grp75 Antibody (PB9642) 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 Grp75 using anti-Grp75 antibody (PB9642).
Grp75 was detected in an immunocytochemical section of A549 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-Grp75 Antibody (PB9642) 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.

Flow Cytometry analysis of HL-60 cells using anti-Grp75 antibody (PB9642).
Overlay histogram showing HL-60 cells stained with PB9642 (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-Grp75 Antibody (PB9642, 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.