Western blot analysis of extracts from various cell lines using HtrA2/Omi (D20A5) Rabbit mAb.
Western blot analysis of extracts from 293T cells, either mock transfected (-) or transfected with a construct expressing full-length mouse HtrA2/Omi (mHtrA2/Omi; +), using HtrA2/Omi (D20A5) Rabbit mAb.
Western blot analysis of extracts from HeLa cells, untreated (upper) or treated with Staurosporine #9953 (1 μM, 3 hr; lower), using HtrA2/Omi (D20A5) Rabbit mAb #9745. Cells were fractionated into whole cell lysate (WCL), cytoplasm (Cyto), membrane (Mem), and cytoskeletal/nucleus (Nuc) using Cell Fractionation Kit #9038. Membrane fraction includes mitochondria.
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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 nonfat dry milk, 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.
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 June 2016
Protocol Id: 263
HtrA2/Omi (D20A5) Rabbit mAb recognizes endogenous levels of total HtrA2/Omi protein. This antibody does not cross-react with HtrA1.Species Reactivity:
Human, Mouse, Rat, Monkey
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Phe341 of human HtrA2/Omi protein.
High temperature requirement protein A2 (HtrA2)/Omi is a serine protease with homology to the E. coli HtrA protein (DegP) and is thought to be involved in apoptosis and stress-induced degradation of misfolded proteins (1). While HtrA2 was orignally identified to be present in either the nucleus (1) or endoplasmic reticulum (2), subsequent studies have shown that it localizes in mitochondria and is released during apoptosis (3-8). HtrA2 is produced as a 50 kDa zymogen that is cleaved to generate a 36 kDa mature protein that exposes an amino terminal motif (AVPS) resembling that of the IAP inhibitor Smac/Diablo (3-8). Like Smac, interaction between HtrA2 and IAP family members, such as XIAP, antagonizes their inhibition of caspase activity and protection from apoptosis (3-8). Interestingly, HtrA2 knock-out mice did not show signs of reduced apoptosis, but rather had a loss of neurons in the striatum and a Parkinson's-like phenotype, suggesting that HtrA2 might have a neuroprotective function (9-11). This activity is associated with the protease activity of HtrA2 (9). Furthermore, research studies have shown that loss of function mutations in the HtrA2 gene are associated with Parkinson's disease (12).
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