Anti-HIF-1 alpha/HIF1A Antibody Picoband®

Western blot analysis of HIF-1 alpha/HIF1A using anti-HIF-1 alpha/HIF1A antibody (A00013-3).
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 30ug of sample under reducing conditions.
Lane 1: human A431 whole cell lysates,
Lane 2: human HEPG2 whole cell lysates,
Lane 3: human U20S whole cell lysates,
Lane 4: rat PC-12 whole cell lysates,
Lane 5: mouse thymus tissue lysates.
After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA 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-HIF-1 alpha/HIF1A antigen affinity purified polyclonal antibody (Catalog # A00013-3) 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 HIF-1 alpha/HIF1A at approximately 93KD. The expected band size for HIF-1 alpha/HIF1A is at 93KD.

IHC analysis of HIF-1 alpha/HIF1A using anti-HIF-1 alpha/HIF1A antibody (A00013-3).
HIF-1 alpha/HIF1A was detected in paraffin-embedded section of human mammary cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2μg/ml rabbit anti-HIF-1 alpha/HIF1A Antibody (A00013-3) 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 HIF-1 alpha/HIF1A using anti-HIF-1 alpha/HIF1A antibody (A00013-3).
HIF-1 alpha/HIF1A was detected in paraffin-embedded section of human rectal cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2μg/ml rabbit anti-HIF-1 alpha/HIF1A Antibody (A00013-3) 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 HIF-1 alpha/HIF1A using anti-HIF-1 alpha/HIF1A antibody (A00013-3).
HIF-1 alpha/HIF1A was detected in paraffin-embedded section of mouse intestine tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2μg/ml rabbit anti-HIF-1 alpha/HIF1A Antibody (A00013-3) 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 HIF-1 alpha/HIF1A using anti-HIF-1 alpha/HIF1A antibody (A00013-3).
HIF-1 alpha/HIF1A was detected in paraffin-embedded section of rat intestine tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2μg/ml rabbit anti-HIF-1 alpha/HIF1A Antibody (A00013-3) 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.

Flow Cytometry analysis of A431 cells using anti-HIF-1 alpha/HIF1A antibody (A00013-3).
Overlay histogram showing A431 cells stained with A00013-3 (Blue line).The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-HIF-1 alpha/HIF1A Antibody (A00013-3, 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 (Red line) was also used as a control.