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Western blot analysis of extracts from various cell lines using UBE2C Antibody (upper) and GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).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 UBE2C protein (hUBE2C-Myc/DDK; +), using UBE2C 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.
UBE2C Antibody recognizes endogenous levels of total UBE2C protein.
Human, Mouse, Rat, Monkey
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human UBE2C 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).
Ubiquitin-conjugating enzyme 2C (UBE2C) is one of several ubiquitin conjugating enzymes participating in the E3 anaphase-promoting complex (APC/C). UBE2C is involved in the control of multiple stages of the cell cycle including inactivation of the mitotic spindle assembly checkpoint (2). UBE2C facilitates ubiquitin-dependent proteasomal degradation by initiating K11-linked ubiquitin chains on APC/C substrates (3). Research studies show that UBE2C expression is low in normal tissues, but its expression is dramatically upregulated in tumors derived from tissues such as lung, breast, and prostate (3-8). Overexpression of UBE2C in many types of solid tumors has been attributed to genomic amplification of the UBE2C locus and research studies have suggested that inhibition of UBE2C activity may have therapeutic potential (9).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. XP is a registered trademark of Cell Signaling Technology, Inc. Tween is a registered trademark of ICI Americas, Inc.
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