Anti-HIF-1 alpha antibody [RM1116] - BSA and Azide free

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-HIF-1 alpha antibody [RM1116] - BSA and Azide free (AB317045)

This data was developed using ab317044, the same antibody clone in a different buffer formulation.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HCT116 (human colon epithelial) treated with 0.5mM DFO for 24 hours(Red) / Untreated HCT116 (Red Dotted) cells labelling HIF-1 alpha with ab317044 at 1/5000 dilution (0.01 ug)/Red and Dotted Red (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-HIF-1 alpha antibody [RM1116] - BSA and Azide free (AB317045)

This data was developed using ab317044, the same antibody clone in a different buffer formulation.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HIF1A KO HCT116(HIF1A knockout human colorectal carcinoma epithelial cell),ab255394 cells labelling HIF-1 alpha with ab317044 at 1/200 (2.53 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green).

Confocal image showing negative staining in both HIF1A knockout HCT 116 cells and wildtype HCT 116 cells, the nuclear signal was increased in wildtype HCT 116 cells treated with 0.5 mM DFO for 24 hours (shown in green). The counterstain was observed in red. Nuclear DNA was labelled with DAPI (shown in blue).
Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/ml dilution.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-HIF-1 alpha antibody [RM1116] - BSA and Azide free (AB317045)

This data was developed using ab317044, the same antibody clone in a different buffer formulation.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized bEnd.3 (mouse brain endothelial cell) cells labelling HIF-1 alpha with ab317044 at 1/200 (2.53 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green).

Confocal image showing increased nuclear staining in bEnd.3 cells treated with 0.5 mM DFO for 24 hours (shown in green). The counterstain was observed in red. Nuclear DNA was labelled with DAPI (shown in blue).
Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/ml dilution.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-HIF-1 alpha antibody [RM1116] - BSA and Azide free (AB317045)

This data was developed using ab317044, the same antibody clone in a different buffer formulation.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HeLa (human cervical adenocarcinoma epithelial cell) cells labelling HIF-1 alpha with ab317044 at 1/200 (2.53 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green).

Confocal image showing increased nuclear staining in HeLa cells treated with 1mM DFO for 24 hours (shown in green). The counterstain was observed in red. Nuclear DNA was labelled with DAPI (shown in blue).
Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/ml dilution.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-HIF-1 alpha antibody [RM1116] - BSA and Azide free (AB317045)

This data was developed using ab317044, the same antibody clone in a different buffer formulation.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized bEnd.3 (mouse brain endothelial cell) treated with 0.5mM DFO for 24 hours(Red) / Untreated bEnd.3 (Red Dotted) cells labelling HIF-1 alpha with ab317044 at 1/5000 dilution (0.01 ug)/Red and Dotted Red (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

• WB

Supplier Data

Western blot - Anti-HIF-1 alpha antibody [RM1116] - BSA and Azide free (AB317045)

This data was developed using ab317044, the same antibody clone in a different buffer formulation.

The observed MW is consistent with what has been described in the literature (PMID : 23076220)

The band beneath the target band is likely to be degraded target fragments.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-HIF-1 alpha antibody [RM1116] (<a href='/en-us/products/primary-antibodies/hif-1-alpha-antibody-rm1116-ab317044'>ab317044</a>) 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

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 110 kDa,36 kDa

false

Exposure time: 6s

• WB

Supplier Data

Western blot - Anti-HIF-1 alpha antibody [RM1116] - BSA and Azide free (AB317045)

This data was developed using ab317044, the same antibody clone in a different buffer formulation.

Lysates/proteins at 20 MSD1483 per lane.

In Western blot, ab317044 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) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-HIF-1 alpha antibody [RM1116] (<a href='/en-us/products/primary-antibodies/hif-1-alpha-antibody-rm1116-ab317044'>ab317044</a>) 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 hour 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 hour whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 110 kDa,36 kDa

false

Exposure time: 26s

• WB

Supplier Data

Western blot - Anti-HIF-1 alpha antibody [RM1116] - BSA and Azide free (AB317045)

This data was developed using ab317044, the same antibody clone in a different buffer formulation.

The bands beneath the target band (110 kDa) are likely to be degraded target fragments.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-HIF-1 alpha antibody [RM1116] (<a href='/en-us/products/primary-antibodies/hif-1-alpha-antibody-rm1116-ab317044'>ab317044</a>) at 1/1000 dilution

Lane 1:

Untreated bEnd.3 (mouse brain endothelial cell) whole cell lysate at 20 µg

Lane 2:

bEnd.3 treated with 0.5mM DFO for 24 hours whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 110 kDa,36 kDa

false

Exposure time: 15s