Western blot analysis of extracts from various cell lines using USP4 Antibody.
Western blot analysis of extracts from 293T cells, either mock transfected (-) or transfected with a Myc/DDK-tagged cDNA expression construct encoding full-length human USP4, isoform1/UnpEL (hUSP4-Myc/DDK; +), using USP4 Antibody.
|REACTIVITY||H M R Mk|
|MW (kDa)||104, 109|
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.
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
USP4 Antibody recognizes endogenous levels of total USP4 protein. Based upon sequence alignment, this antibody is predicted to cross-react with the UnpEL and UnpES isoforms of USP4. This antibody does not cross-react with USP15.Species Reactivity:
Human, Mouse, Rat, MonkeySpecies predicted to react based on 100% sequence homology:
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding His666 of human USP4 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. USP4 was originally identified during a survey of murine genes near the Mpv20 retroviral insertion site and intially referred to as Ubiquitous Nuclear Protein (UNP). Analysis of the mouse cDNA originally identified Usp4/Unp as a proto-oncogene related to the human tre-2/tre-17/USP6 proto-oncogene (3,4). Usp4/Unp was subsequently observed to contain the conserved Cys and His boxes of the UBP family (5,6) as well as DUB activity (7,8). In a study of primary lung tumor tissue, it was observed that the human homolog of Usp4, USP4/UNPH, had elevated gene expression levels in small cell tumors and adenocarcinomas of the lung, suggesting a causative role for USP4 in neoplasia (6). Another recent study demonstrated overexpression of USP4 in several types of human cancer and that USP4 positively contributes to cell transformation by negatively regulating p53 levels (9). Both murine and human USP4 have been shown to interact with the Rb family of tumor suppressor proteins, providing additional mechanistic evidence of a role for USP4 in cellular transformation (10, 11).
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