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Recombinant fragment corresponding to Human HIF-1-alpha aa 530-826 (C terminal).
Under normoxic conditions HIF-1 alpha has a short half-life. It is largely undetectable in cells or tissues grown under normoxic conditions. It is stabilized only at O2 concentrations below 5% and upon stabilization under hypoxic conditions HIF-1 translocates to the nucleus. Therefore we recommend western blots using nuclear extracts and running Hypoxia treated samples as positive control (ab180880). Hypoxia can be induced with treatment using certain agents e.g. CoCl2 or DFO, etc. so proper sample preparation is critical.
Our Abpromise guarantee covers the use of ab16066 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|Flow Cyt||Use 2µg for 106 cells.
ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.
|Immunoelectrophoresis||Use at an assay dependent concentration.|
|IHC-P||Use a concentration of 10 µg/ml.|
|EMSA||Use at an assay dependent concentration. PubMed: 21193390|
|Gel supershift assays||Use at an assay dependent concentration. Only been successfully used with mouse HIF1 alpha.|
|IP||Use at an assay dependent concentration.|
|WB||1/2000. Detects a band of approximately 116 kDa (predicted molecular weight: 93 kDa). 116 kDa band represents HIF-1 alpha after hypoxic induction.|
|IHC-P||1/800. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. See Abreview for further information|
IHC image of ab16066 staining HIF-1 alpha in human colon formalin fixed paraffin embedded tissue sections*, performed on a Leica Bond. The section was pre-treated using heat mediated antigen retrieval with Tris/EDTA buffer (pH9, epitope retrieval solution 2) for 20 mins. The section was then incubated with ab16066, 10μg/ml working concentration, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. No primary antibody was used in the secondary only control (shown on the inset).
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre
Immunocytochemistry/Immunofluorescence analysis of U251 cells labeling HIF-1-alpha with ab16066. HIF-1-alpha (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control - right) or with ab16066 (left) at a dilution of 1/20 over night at 4°C and incubated with a DyLight-488 conjugated secondary antibody. Images were taken at 60X magnification.
This image is courtesy of an anonymous Abreview
Overlay histogram showing HeLa cells stained with ab16066 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab16066, 2µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.
Immunocytochemistry/Immunofluorescence analysis of HeLa cells labeling HIF-1-alpha with ab16066. HIF-1 alpha (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control - right) or with ab16066 (left) at a dilution of 1/20 over night at 4°C and incubated with a DyLight-488 conjugated secondary antibody. Images were taken at 60X magnification.
Immunocytochemistry/Immunofluorescent analysis of HIF-1-alpha (green) in HeLa cells either left untreated (left panel) or treated with 100uM Deferoxamine mesylate for ~16 hours (right panel). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 15 minutes at room temperature and blocked with 0.3% BSA for 15 minutes at room temperature. Cells were probed with ab16066 at a dilution of 1/100 for at least 1 hour at room temperature and washed with PBS. Cells were then incubated with a DyLight 488-conjugated goat anti-mouse IgG secondary antibody at a dilution of 1/500 for 30 minutes at room temperature. F-actin (red) was stained with DyLight 594 Phalloidin and nuclei (blue) were stained with Hoechst dye.
ab16066 staining HIF-1-alpha in Human small intestine (IBD) and tonsil tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde; antigen retrieval was by heat mediation with an EDTA buffer (pH 9.0). Samples were incubated with primary antibody (1/800 in diluent + background reducers) for 20 minutess at 25°C. An undiluted Goat polymer was used as the secondary antibody.
Immunocytochemistry/Immunofluorescence analysis of A2058 cells labeling HIF-1-alpha with ab16066. HIF-1-alpha (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control - right) or with ab16066 (left) at a dilution of 1/20 over night at 4°C and incubated with a DyLight-488 conjugated secondary antibody. Images were taken at 60X magnification.
Immunocytochemistry/Immunofluorescence analysis of Human fibroblasts labeling HIF-1-alpha (green) with ab16066. Cells were fixed with paraformaldehyde, permeabilized with 0.3% Triton X-100 and blocked with 5% serum for 1 hour at 24°C. Samples were incubated with primary antibody (1/500 in 0.3% Triton X-100 + 1% BSA) for 1 hour 30 minutes at 24°C. An Alexa Fluor® 488-conjugated goat anti-mouse polyclonal IgG (1/2000) was used as the secondary antibody.
Immunohistochemistry was performed on normal biopsies of deparaffinized Human tonsil tissue. To expose target proteins heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1/20 with ab16066 (left) or without primary antibody (negative control - right) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.
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