Western blot analysis of extracts from U-2 OS cells, untreated (-) or treated (+) with MG132 (10 µM, 8 hr), using TCF11/NRF1 (D5B10) Rabbit mAb and β-Actin (D6A8) Rabbit mAb #8457.
|REACTIVITY||H M Mk|
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
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
TCF11/NRF1 (D5B10) Rabbit mAb recognizes endogenous levels of total TCF11 protein.Species Reactivity:
Human, Mouse, Monkey
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Gly129 of human TCF11 protein.
Transcription factor 11 (TCF11) is a basic leucine zipper transcription factor. It is also referred to as Nuclear factor E2-related factor 1 (NRF1). TCF11 was initially reported to activate erythroid-specific, human globin gene expression (1). It plays an essential role during embryonic development (2). It also associates with other transcription factors, such as Jun proteins, to transcriptionally control antioxidant response element (ARE)-mediated expression in response to antioxidants and xenobiotics (3-5). TCF11 has been shown to regulate proteasomal degradation and mediate the proteasome recovery pathway after proteasome inhibition (6,7). TCF11 is ubiquitously expressed (8) and several isoforms have been reported. The 120 kDa form exists in the endoplasmic reticulum (ER) membrane under normal conditions. Upon proteasome inhibition, TCF11 translocates to the nucleus (9). The 65 kDa N-terminal-truncated form is constitutively localized to the nucleus (10,11). TCF11 protein levels are regulated by ubiquitination and proteasomal-mediated degradation (12); proteasome inhibitors stabilize TCF11.
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