Western blot analysis of extracts from various cell lines using UBE2O Antibody.Learn more about how we get our images.
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing Myc/DDK-tagged full-length human UBE2O protein (hUBE2O-Myc/DDK; +), using UBE2O Antibody (upper) and DYKDDDDK Tag Antibody #2368 (lower).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.
UBE2O Antibody recognizes endogenous levels of total UBE2O protein.
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Val576 of human UBE2O protein. Antibodies are purified by protein A and peptide affinity chromatography.
Protein ubiquitination requires the concerted action of the E1, E2, and E3 ubiquitin-conjugating enzymes. Ubiquitin is first activated through ATP-dependent formation of a thiol ester with ubiquitin-activating enzyme E1. The activated ubiquitin is then transferred to a thiol group of ubiquitin-carrier enzyme E2. The final step is the transfer of ubiquitin from E2 to an ε-amino group of the target protein lysine residue, which is mediated by ubiquitin-ligase enzyme E3 (1).
UBE2O (E2-230K) is a unique member of the E2 family of ubiquitin-conjugating enzymes in that it functions as an E2-E3 hybrid enzyme (2). Research studies have demonstrated that UBE2O expression is regulated during erythroid differentiation, which suggests that its enzymatic activity participates in shaping the architecture of the erythrocyte proteome (3). UBE2O has also been implicated in BMP-7-induced adipocyte differentiation through its regulation of SMAD6 monoubiquitination (4).
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