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Western blot analysis of extracts from various cell lines using USP2 Antibody.Learn more about how we get our images.
Western blot analysis of extracts from COS-7 cells, either mock transfected (-) or transfected with a Myc/DDK-tagged cDNA expression construct encoding full-length transcript variant 1 of human USP2 (+), using USP2 Antibody.Learn more about how we get our images.
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
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C. Do not aliquot the antibody.
USP2 Antibody recognizes endogenous levels of total USP2 protein. This antibody cross-reacts with all known USP2 splice variants but does not cross-react with USP21.
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Leu387 of human USP2 protein. Antibodies are purified by protein A and peptide affinity chromatography.
Ubiquitinating enzymes (UBEs) catalyze protein ubiquitination, a reversible process countered by deubiquitinating enzyme (DUB) action (1,2). Five DUB subfamilies are recognized, including the USP, UCH, OTU, MJD and JAMM enzymes. Ubiquitin-specific-processing protease 2 (USP2) belongs to the USP (UBP/UCH type 2) subfamily and is characterized by its C19 peptidase activity, which is involved in ubiquitin recycling and in the disassembly of various forms of polymeric ubiquitin and ubiquitin-like protein complexes (3). Characteristic of the USP subfamily, USP2 possesses a highly conserved "Cys box" and "His box," which contain a conserved cysteine and histidine residue, respectively, and form part of the active site of this thiol protease. The catalytic core, which lies between the Cys box and His box, is responsible for the deubiquitinating activity of USP2 and is present within each of its splice variants (4,5).
There is mounting evidence that USP2 functions as an oncoprotein through multiple mechanisms. In human prostate cancer, USP2 is highly overexpressed and drives tumor growth by binding to and stabilizing fatty acid synthase through deubiquitination (6,7). It has also been demonstrated that USP2 can bind and deubiquitinate both Mdm2 (8) and cyclin D1 (9), which leads to their stabilization and enhanced cell proliferation.
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