Anti-HIF-1 alpha antibody [SIMA343B]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-HIF-1 alpha antibody [SIMA343B] (AB325355)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HeLa (human cervical adenocarcinoma epithelial cell) and HeLa treated with CoCl2 (0.5 mM) for 6 hr cells labelling HIF-1 alpha with ab325355 at 1/50 dilution, followed by ab150157 Goat Anti-Rat IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution (Green).

Confocal image showing increased nuclear staining in HeLa cells (shown in green) treated with CoCl2 (0.5 mM) for 6 hr. The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab202272 Recombinant Alexa Fluor® 594 Anti-alpha Tubulin antibody [EP1332Y] - Microtubule Marker (ab202272) was used to counterstain tubulin(Magenta). The Nuclear counterstain was DAPI (Blue).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-HIF-1 alpha antibody [SIMA343B] (AB325355)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 (mouse embryonic fibroblast) and NIH/3T3 cells treated with DFO (0.5 mM) for 24 hr and with MG-132 (10 µM) for the last 4 hr cells labelling HIF-1 alpha with ab325355 at 1/50 dilution, followed by ab150157 Goat Anti-Rat IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution (Green).

Confocal image showing increased nuclear staining in NIH/3T3 cells (shown in green) treated with DFO (0.5 mM) for 24 hr and with MG-132 (10 µM) for the last 4 hr. The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab202272 Recombinant Alexa Fluor® 594 Anti-alpha Tubulin antibody [EP1332Y] - Microtubule Marker (ab202272) was used to counterstain tubulin (Magenta). The Nuclear counterstain was DAPI (Blue).

• WB

Lab

Western blot - Anti-HIF-1 alpha antibody [SIMA343B] (AB325355)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

In Western blot, ab325355 was shown to bind specifically to HIF-1 alpha. A band was observed at 110 kDa in wild-type HCT 116 cell lysates with whereas no signal observed at this size in HIF-1 alpha knockout cell line ab255394.

Lysates were freshly made and used for Western blotting immediately to minimize protein degradation.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-HIF-1 alpha antibody [SIMA343B] (ab325355) at 1/1000 dilution

Lane 1:

Untreated HCT 116 (human colorectal carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

HCT116 treated with 0.5 mM DFO for 24 hours whole cell lysate at 20 µg

Lane 3:

Untreated HIF-1 alpha knockout HCT116 whole cell lysate at 20 µg

Lane 4:

HIF-1 alpha knockout HCT116 treated with 0.5 mM DFO for 24 hours whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rat IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rat-igg-h-l-hrp-ab205720'>ab205720</a>) at 1/10000 dilution

Observed band size: 110 kDa,36 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-HIF-1 alpha antibody [SIMA343B] (AB325355)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-HIF-1 alpha antibody [SIMA343B] (ab325355) at 1/1000 dilution

Lane 1:

Untreated HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

HeLa treated with 0.5mM CoCl2 for 6 hours whole cell lysate at 20 µg

Lane 3:

Untreated HeLa whole cell lysate at 20 µg

Lane 4:

HeLa treated with 0.5mM DFO for 24 hours whole cell lysate at 20 µg

Lane 5:

Untreated NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

Lane 6:

NIH/3T3 treated with 100µM CoCl2 for 4 hours whole cell lysate at 20 µg

Lane 7:

Untreated NIH/3T3 whole cell lysate at 20 µg

Lane 8:

NIH/3T3 treated with 0.5mM CoCl2 for 24 hours, 10µM MG-132 was then added for last 4 hours whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rat IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rat-igg-h-l-hrp-ab205720'>ab205720</a>) at 1/10000 dilution

Observed band size: 110 kDa,36 kDa

false

Exposure time: 37s

• WB

Lab

Western blot - Anti-HIF-1 alpha antibody [SIMA343B] (AB325355)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

In Western blot, Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842) (1 : 100000) (15Kda).

All lanes:

Western blot - Anti-HIF-1 alpha antibody [SIMA343B] (ab325355) at 1/1000 dilution

Lane 1:

293T (human embryonic kidney epithelial cell) cell transfected with an empty vector containing a myc-his-tag, whole cell lysate at 20 µg

Lane 2:

293T cells transfected with a human HIF-1 alpha expression vector containing a myc-his-tag, whole cell lysate at 20 µg

Lane 3:

293T cells transfected with a human EPAS1 expression vector containing a myc-his-tag, whole cell lysate at 20 µg

Lane 4:

293T cell transfected with an empty vector containing a myc-his-tag, whole cell lysate at 20 µg

Lane 5:

293T cells transfected with a mouse HIF-1 alpha expression vector containing a myc-his-tag, whole cell lysate at 20 µg

Lane 6:

293T cells transfected with a mouse EPAS1 expression vector containing a myc-his-tag, whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rat IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rat-igg-h-l-hrp-ab205720'>ab205720</a>) at 1/10000 dilution

Observed band size: 110 kDa,15 kDa

false

Exposure time: 1s