Western blot analysis of extracts from various cell types using FIH (D19B3) Rabbit mAb.
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Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody datasheet or product webpage for recommended antibody dilution.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised October 2016
Protocol Id: 10
FIH (D19B3) Rabbit mAb detects endogenous levels of total FIH protein.Species Reactivity:
Human, Mouse, Rat, Monkey
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Tyr35 of human FIH protein.
FIH (Factor inhibiting HIF-1, HIF asparagine hydroxylase) is a dioxygen-dependent asparaginyl hydroxylase that modifies target protein function by hydroxylating target protein asparagine residues (1-3). Hypoxia-inducible factor (HIF), a transcriptional activator involved in control of cell cycle in response to hypoxic conditions, is an important target for FIH regulation. FIH functions as an oxygen sensor that regulates HIF function by hydroxylating at Asn803 in the carboxy-terminal transactivation domain (CAD) of HIF (4,5). During normoxia, FIH uses cellular oxygen to hydroxylate HIF-1 and prevent interaction of HIF-1 with transcriptional coactivators, including the CBP/p300-interacting transactivator. Under hypoxic conditions, FIH remains inactive and does not inhibit HIF, allowing the activator to regulate transcription of genes in response to low oxygen conditions (4-6). FIH activity is regulated in through interaction with proteins, including Siah-1, which targets FIH for proteasomal degradation (7). The Cut-like homeodomain protein CDP can bind the FIH promoter region to regulate FIH expression at the transcriptional level (8). Phosphorylation of HIF at Thr796 also can prevent FIH hydroxylation on Asn803 (9). Potential FIH substrates also include proteins with ankyrin repeat domains, such as Iκ-B, Notch, and ASB4 (10-12).
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