Western blot analysis of extracts from various cell lines using TCEB3/Elongin A Antibody.
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
TCEB3/Elongin A Antibody recognizes endogenous levels of total TCEB3/Elongin A protein.Species Reactivity:
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human TCEB3/Elongin A protein. Antibodies are purified by protein A and peptide affinity chromatography.
The Elongin complex is a heterotrimer composed of TCEB3/Elongin A, Elongin B (TCEB2), and Elongin C (TCEB1) subunits (1-3). The Elongin complex regulates the rate of RNA polymerase II (RNAPII) transcription elongation by releasing the transient pausing of RNAPII at multiple sites along the DNA. TCEB3/Elongin A is the transcriptionally active subunit, while Elongin B and C subunits play a regulatory role (3,4). TCEB3/Elongin A may be required for expression of a subset of cell cycle regulated genes, and embryonic stem (ES) cells lacking TCEB3/Elongin A show abnormalities in cell size, growth, and cell cycle distribution (5). In addition, the Elongin complex has been shown to interact with the cullin family and RING finger proteins Cul5/Rbx2 upon UV-induced DNA damage, removing arrested RNAPII at sites of DNA damage by ubiquitination and degradation as part of an E3 ubiquitin ligase (6).
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