Western blot analysis of cell lysates from HT1080, HT1080 BRF1-/-, HCT15, C6, COS and NIH/3T3 cells, using BRF1/2 Antibody.
|REACTIVITY||H M R Mk|
|MW (kDa)||40 to 50, 62|
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 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
This antibody detects endogenous levels of total BRF1 and BRF2 proteins.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 the carboxy terminus of human BRF1 protein. Antibodies are purified by protein A and peptide affinity chromatography.
Butyrate response factor 1 (BRF1; also known as EGF response factor 1 [ERF1], TIS11B, ZFP36L1) and butyrate response factor 2 (BRF2; also known as EGF response factor 2 [ERF2], TIS11D, ZFP36L2) both belong to the TIS11 family of CCCH zinc-finger proteins (1). This family of proteins, which also includes tristetraprolin (TTP), bind to AU-rich elements (ARE) found in the 3'-untranslated regions of mRNAs and promote de-adenylation and rapid degradation by the exosome (2,3). These proteins play a critical role in cell growth control by regulating the mRNA turnover of multiple cytokines, growth factors and cell cycle regulators, including GM-CSF, TNFα, IL-2, IL-3 and IL-6 (4,5). Deregulated ARE-mRNA stability can contribute to both inflammation and oncogenic transformation (6-8). Insulin-induced stabilization of ARE-containing transcripts is mediated by Akt/PKB phosphorylation of BRF1 at Ser92, which results in binding by 14-3-3 protein and inactivation of BRF1 (9).
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