Anti-HIF-1 alpha antibody [H1alpha67]

7 product images

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  Flow Cytometry (Intracellular) - Anti-HIF-1 alpha antibody [H1alpha67] (ab1)

  Flow cytometry using ab1. HeLa (Human epithelial cell line from cervix adenocarcinoma) cells were cultured untreated or with 1mM Deferoxamine (Deferoxamine mesylate, Iron chelator ab120727) for 24 hours to induce HIF-1-alpha protein levels. Cells were then trypsinized, fixed with paraformaldehyde and stained with ab1 (0.5 μg/mL). 1% BSA in PBS was used as the blocking buffer throughout. ab1 was labeled with and anti-mouse Alexa-Fluor^® 488 dye. Unstained (black), untreated (red) and DFO treated (blue) cell traces are shown.

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  Immunoprecipitation - Anti-HIF-1 alpha antibody [H1alpha67] (ab1)

  HIF-1 alpha was immunoprecipitated using 0.5 mg HeLa Nuclear DFO treated whole cell extract (Hela-DFO treated (0.5mM, 24h) Nuclear Lysate ab180880), 5 μg of Mouse monoclonal to HIF-1 alpha and 50 μl of protein G magnetic beads (+). No antibody was added to the control (-).
  

  The antibody was incubated under agitation with Protein G beads for 10 minutes, HeLa DFO treated whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10 minutes under agitation.

  Proteins were eluted by addition of 40 μl SDS loading buffer and incubated for 10 minutes at 70°C; 10 μl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab1.

  Secondary: Goat polyclonal to mouse IgG light chain specific (HRP) at 1:20,000 dilution.

  Band: 110 kDa; HIF1 alpha

  All lanes: Immunoprecipitation - Anti-HIF-1 alpha antibody [H1alpha67] (ab1)

  Developed using the ECL technique.

  Performed under reducing conditions.

  Predicted band size: 92 kDa

  Exposure time: 20min

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  Western blot - Anti-HIF-1 alpha antibody [H1alpha67] (ab1)

  All lanes: Western blot - Anti-HIF-1 alpha antibody [H1alpha67] (ab1)

  All lanes: Hela cell lysate

  Secondary

  All lanes: Western blot

  Predicted band size: 56 kDa

  Observed band size: 23 kDa

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  Western blot - Anti-HIF-1 alpha antibody [H1alpha67] (ab1)

  We recommend using 5% milk in TBST as the blocking agent, decreasing to 2% milk in TBST during primary and secondary antibody incubation.
  

  Blots were developed with Goat Anti-Mouse IgG H&L (HRP) preadsorbed (Goat Anti-Mouse IgG H&L (HRP) preadsorbed ab97040) secondary antibody

  All lanes: Western blot - Anti-HIF-1 alpha antibody [H1alpha67] (ab1) at 5 µg/mL

  Lane 1: Western blot - HeLa nuclear extract lysate (HeLa nuclear extract lysate ab150036) at 40 µg

  Lane 2: Western blot - Hela-DFO treated (0.5mM, 24h) Nuclear Lysate (Hela-DFO treated (0.5mM, 24h) Nuclear Lysate ab180880) at 40 µg

  Lane 3: HeLa nuclear control at 40 µg

  Lane 4: HeLa nuclear DFO treated at 40 µg

  Lane 5: Western blot - Recombinant Human HIF-1 alpha protein (Recombinant Human HIF-1 alpha protein ab154478) at 0.001 µg

  Secondary

  All lanes: Western blot - Goat Anti-Mouse IgG H&L (HRP) preadsorbed (Goat Anti-Mouse IgG H&L (HRP) preadsorbed ab97040) at 1/10000 dilution

  Performed under reducing conditions.

  Predicted band size: 92 kDa

  Exposure time: 20min

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  This image is taken from a customer review submitted by Mike Campa, no further information is known about this image

  Western blot - Anti-HIF-1 alpha antibody [H1alpha67] (ab1)

  PVDF membrane was used and blocked for 16 hours in 5% milk.

  All lanes: Western blot - Anti-HIF-1 alpha antibody [H1alpha67] (ab1) at 1/400 dilution

  All lanes: Human whole cell lysate (human lung adenocarcinoma cell line ADLC-5M2) treated for 16 hours with 100 micromolar deferoxamine (DFO) at 20 µg

  Performed under reducing conditions.

  Predicted band size: 14 kDa, 54 kDa, 92 kDa

  Observed band size: 120 kDa

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  Immunocytochemistry/ Immunofluorescence - Anti-HIF-1 alpha antibody [H1alpha67] (ab1)

  ab1 staining HIF-1 alpha in HeLa DFO cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab1 at 10µg/ml and Anti-beta Tubulin antibody - Loading Control ab6046, Rabbit polyclonal to beta Tubulin - Loading Control. Cells were then incubated with Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed ab150117, Goat polyclonal Secondary Antibody to Mouse IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (shown in green) and Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) ab150080, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 594) at 1/1000 dilution (shown in pseudocolour magenta). Nuclear DNA was labelled with DAPI (shown in blue).

  Also suitable in cells fixed with 4% paraformaldehyde (10 min).

  Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

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  Western blot - Anti-HIF-1 alpha antibody [H1alpha67] (ab1)

  Western blot: Anti-HIF1A antibody [H1alpha67] (ab1) staining at 5 ug/ml, shown in green; Rabbit anti-alpha Tubulin antibody [EP1332Y] (Anti-alpha Tubulin antibody [EP1332Y] - Microtubule Marker ab52866) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab1 was shown to bind specifically to HIF1A. A band was observed at 100 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in HIF1A knockout cell line. To generate this image, wild-type and HIF1A knockout HCT 116 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Mouse IgG H&L 800CW and Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution.

  All lanes: Western blot - Anti-HIF-1 alpha antibody [H1alpha67] (ab1) at 5 µg/mL

  Lane 1: Wild-type HCT 116 DMOG (0 mM, 4 h) nuclear cell lysate at 20 µg

  Lane 2: Wild-type HCT 116 treated DMOG (1 mM, 4 h) nuclear cell lysate at 20 µg

  Lane 3: HIF1A knockout HCT 116 DMOG (0 mM, 4 h) nuclear cell lysate at 20 µg

  Lane 4: HIF1A knockout HCT 116 treated DMOG (1 mM, 4 h) nuclear cell lysate at 20 µg

  Lane 5: Wild-type HCT 116 DMOG (0 mM, 4 h) cell lysate at 20 µg

  Lane 6: Wild-type HCT 116 treated DMOG (1 mM, 4 h) cell lysate at 20 µg

  Lane 7: HIF1A knockout HCT 116 DMOG (0 mM, 4 h) cell lysate at 20 µg

  Lane 8: HIF1A knockout HCT 116 treated DMOG (1 mM, 4 h) cell lysate at 20 µg

  Secondary

  All lanes: Goat anti-Mouse IgG H&L 800CW and Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution

  Performed under reducing conditions.

  Observed band size: 100 kDa