Anti-HSPB8/Hsp22 Antibody Picoband®

Western blot analysis of HSPB8/Hsp22 using anti-HSPB8/Hsp22 antibody (A02492-2).
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 MCF-7 whole cell lysates,
Lane 2: human U87 whole cell lysates,
Lane 3: rat H9C2 whole cell lysates,
Lane 4: rat heart tissue lysates,
Lane 5: mouse C2C12 whole cell lysates,
Lane 6: mouse skeletal muscle tissue lysates,
Lane 7: mouse heart 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-HSPB8/Hsp22 antigen affinity purified polyclonal antibody (Catalog # A02492-2) 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 HSPB8/Hsp22 at approximately 22 kDa. The expected band size for HSPB8/Hsp22 is at 22 kDa.

IHC analysis of HSPB8/Hsp22 using anti-HSPB8/Hsp22 antibody (A02492-2).
HSPB8/Hsp22 was detected in paraffin-embedded section of mouse pancreas tissues. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-HSPB8/Hsp22 Antibody (A02492-2) overnight at 4°C. Biotinylated goat anti-rabbit 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 # SA1022) with DAB as the chromogen.

IHC analysis of HSPB8/Hsp22 using anti-HSPB8/Hsp22 antibody (A02492-2).
HSPB8/Hsp22 was detected in paraffin-embedded section of rat pancreas tissues. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-HSPB8/Hsp22 Antibody (A02492-2) overnight at 4°C. Biotinylated goat anti-rabbit 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 # SA1022) with DAB as the chromogen.

IHC analysis of HSPB8/Hsp22 using anti-HSPB8/Hsp22 antibody (A02492-2).
HSPB8/Hsp22 was detected in paraffin-embedded section of human lung cancer tissues. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-HSPB8/Hsp22 Antibody (A02492-2) overnight at 4°C. Biotinylated goat anti-rabbit 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 # SA1022) with DAB as the chromogen.

IHC analysis of HSPB8/Hsp22 using anti-HSPB8/Hsp22 antibody (A02492-2).
HSPB8/Hsp22 was detected in paraffin-embedded section of human mammary cancer tissues. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-HSPB8/Hsp22 Antibody (A02492-2) overnight at 4°C. Biotinylated goat anti-rabbit 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 # SA1022) with DAB as the chromogen.

IF analysis of HSPB8/Hsp22 using anti-HSPB8/Hsp22 antibody (A02492-2).
HSPB8/Hsp22 was detected in immunocytochemical section of MCF7 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 2μg/mL rabbit anti-HSPB8/Hsp22 Antibody (A02492-2) overnight at 4°C. DyLight®594 Conjugated Goat Anti-Rabbit IgG (BA1142) was used as secondary antibody at 1:100 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 U2OS cells using anti-HSPB8/Hsp22 antibody (A02492-2).
Overlay histogram showing U2OS cells stained with A02492-2 (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-HSPB8/Hsp22 Antibody (A02492-2, 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.

Immunoprecipitating HSPB8/Hsp22 in MCF-7 whole cell lysate.
Western blot analysis of HSPB8/Hsp22 using anti-HSPB8/Hsp22 antibody (A02492-2);
Lane 1: MCF-7 whole cell lysates (30ug);
Lane 2: Rabbit control IgG instead of anti-HSPB8/Hsp22 antibody in MCF-7 whole cell lysate;
Lane 3: anti-HSPB8/Hsp22 antibody (2μg) + MCF-7 whole cell lysate (500μg).
After electrophoresis, proteins were transferred to a membrane. Then the membrane was incubated with rabbit anti-HSPB8/Hsp22 antigen affinity purified polyclonal antibody (A02492-2) 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 HSPB8/Hsp22 at approximately 22 kDa. The expected band size for HSPB8/Hsp22 is at 22 kDa.