Anti-HIF-1 alpha 抗体 [H1alpha67]

• ICC/IF

Lab

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 ab6046, Rabbit polyclonal to beta Tubulin - Loading Control. Cells were then incubated with ab150117, Goat polyclonal Secondary Antibody to Mouse IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (shown in green) and 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.

• Flow Cyt (Intra)

Characteriser

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 (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.

• IP

Lab

Immunoprecipitation - Anti-HIF-1 alpha antibody [H1alpha67] (AB1)

HIF-1 alpha was immunoprecipitated using 0.5 mg HeLa Nuclear DFO treated whole cell extract (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)

Predicted band size: 92 kDa

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Exposure time: 20min

• WB

AbReview4603****

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

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

Observed band size: 120 kDa

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This image is taken from an Abreview submitted by Mike Campa, no further information is known about this image

• WB

Lab

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 (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 (<a href='/products/tissue-lysates/hela-nuclear-extract-lysate-ab150036'>ab150036</a>) at 40 µg

Lane 2:

Western blot - Hela-DFO treated (0.5mM, 24h) Nuclear Lysate (<a href='/products/tissue-lysates/hela-dfo-treated-05mm-24h-nuclear-lysate-ab180880'>ab180880</a>) 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 (<a href='/products/proteins-peptides/recombinant-human-hif-1-alpha-protein-ab154478'>ab154478</a>) at 0.001 µg

Secondary

All lanes:

Western blot - Goat Anti-Mouse IgG H&L (HRP) preadsorbed (<a href='/products/secondary-antibodies/goat-mouse-igg-h-l-hrp-preadsorbed-ab97040'>ab97040</a>) at 1/10000 dilution

Predicted band size: 92 kDa

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Exposure time: 20min

• WB

Lab

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] (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

Observed band size: 100 kDa

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• WB

Ap254****

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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