Anti-Hsp27/HSPB1 Antibody Picoband®

Western blot analysis of HSP27 using anti-HSP27 antibody (PB9237).
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 A549 whole cell lysates,
Lane 2: human CACO-2 whole cell lysates,
Lane 3: human Hela whole cell lysates,
Lane 4: human MCF-7 whole cell lysates,
Lane 5: human HT1080 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-HSP27 antigen affinity purified polyclonal antibody (Catalog # PB9237) 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 HSP27 at approximately 27 kDa. The expected band size for HSP27 is at 27 kDa.

IHC analysis of HSP27 using anti-HSP27 antibody (PB9237).
HSP27 was detected in paraffin-embedded section of human intestinal cancer tissue. 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-HSP27 Antibody (PB9237) 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 HSP27 using anti-HSP27 antibody (PB9237).
HSP27 was detected in paraffin-embedded section of human mammary cancer tissue. 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-HSP27 Antibody (PB9237) 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 HSP27 using anti-HSP27 antibody (PB9237).
HSP27 was detected in frozen section of human placenta tissue. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-HSP27 Antibody (PB9237) 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 HSP27 using anti-HSP27 antibody (PB9237).
HSP27 was detected in immunocytochemical section of U20S 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-HSP27 Antibody (PB9237) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Rabbit IgG (BA1127) 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 A431 cells using anti-HSP27 antibody (PB9237).
Overlay histogram showing A431 cells stained with PB9237 (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-HSP27 Antibody (PB9237,1μg/1x10⁶ cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-rabbit IgG (BA1127, 5-10μg/1x10⁶ cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was rabbit 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.

Immunoprecipitating HSP27 in Hela whole cell lysate.
Western blot analysis of HSP27 using anti-HSP27 antibody (PB9237).
Lane 1: Hela whole cell lysates (30ug),
Lane 2: Rabbit control IgG instead of anti-HSP27 antibody in Hela whole cell lysate,
Lane 3: anti-HSP27 antibody (2μg) + Hela whole cell lysate (500μg).
After electrophoresis, proteins were transferred to a membrane. Then the membrane was incubated with rabbit anti-HSP27 antigen affinity purified polyclonal antibody (PB9237) at a dilution of 0.5 μg/mL and probed with a mouse anti-rabbit IgG-HRP secondary antibody. The signal is developed using ECL Plus Western Blotting Substrate (Catalog # AR1196-200). A specific band was detected for HSP27 at approximately 27 kDa. The expected band size for HSP27 is at 27 kDa.