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Our Abpromise guarantee covers the use of ab199 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 at an assay dependent concentration.|
|IHC-Fr||Use at an assay dependent concentration.|
|ChIP||Use at an assay dependent concentration. PubMed: 19897487|
|IP||Use at 5-1 µg/mg of lysate.|
|ICC/IF||Use a concentration of 1 µg/ml.|
|EMSA||Use at an assay dependent concentration.|
|WB||1/200 - 1/1000. Detects a band of approximately 118 kDa (predicted molecular weight: 100 kDa). The following should be taken into consideration: sufficient hypoxia induction is necessary; use nuclear extracts for cleaner results; use positive/negative controls to see which band is up-regulated; HIF2a degrades rapidly (fast sample preparation, protease inhibitors!).|
Analysis on normoxic and hypoxic nuclear rat cell lysates.
ab199 staining human adrenal tissue sections by IHC-P. Sections were PFA fixed and subjected to heat mediated antigen retrieval in citrate buffer (pH 6) prior to blocking in 10% serum for 10 minutes at 25°C. The primary anitbody was diluted 1/500 and incubated with the sample for 1 hour at 25°C. A biotinylated goat anti-rabbit antibody was used as the secondary.
Image from PLoS One. 2012; 7(1): e29876. Fig3A, doi: 10.1371/journal.pone.0029876
HIF-2α levels in HIF-2α−/− and WT mouse kidneys.
HIF-2α−/− mice or their wild-type littermates were exposed to left ureteral obstruction (UO), which continued for 24 hours, and then was released. 2 days after release of UO or at the corresponding time point in the non-UO sham-operated mice, the left kidneys were harvested and subjected to immunoblot analyses of HIF-2α and co-detection of TBP as a loading control
Nuclear extracts were isolated from harvested whole kidneys using NE-PER Nuclear and Cytoplasmic Extraction Reagents supplemented with Complete Protease Inhibitor Cocktail Tablets. Nuclear protein fractions were electrophoresed on 10% SDS-PAGE under reducing conditions and transferred to a nitrocellulose membrane. Membranes were blocked with LI-COR blocking buffer (LI-COR Biosciences, Lincoln, NE). Membranes were then incubated with the same blocking solution containing rabbit polyclonal primary antibodies against HIF-2α (1:500, ab199). After washing, membranes were incubated at room temperature for 1 h in TBS/0.05% Tween 20 buffer with the IRDye800 secondary antibodies (1:10000) and then washed again in TBS/0.05% Tween 20 for 3 times. The blot was visualized using an Odyssey infrared imaging system. All values were normalized to a loading control TATA binding protein (TBP, 1:2000, ab818, Abcam) and expressed as fold increase relative to control.
ICC/IF image of ab199 stained HepG2 cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab199, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.
This image is courtesy of Marilo Dolores ChiaraThe blots were probed with anti-alpha tubulin as a loading control.