Western blot analysis of extracts from LNCaP, DLD-1, and HT-29 cells using CoREST (D6I2U) Rabbit mAb.
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
CoREST (D6I2U) Rabbit mAb recognizes endogenous levels of total CoREST protein.Species Reactivity:
Monoclonal antibody is produced by immunizing animals with recombinant protein specific to the carboxy terminus of human CoREST protein.
The REST corepressor 1 (CoREST, RCOR1) was first identified as a repressor element 1-silencing transcription factor (REST) corepressor (1,2). The CoREST protein is encoded by the RCOR1 gene and is part of a large, multi-subunit repressor complex that includes the histone demethylase LSD1 and histone deacetylases (HDAC) 1 and 2 (1,3-5). CoREST binds the carboxy-terminal domain of REST and is recruited to repress neuronal gene transcription in non-neuronal and neural stem cells (1,6,7). The REST corepressor is essential for repressor complex-nucleosome interaction, the subsequent deacetylation of histone amino-terminal tails by HDAC1/2, and the LSD1 methylation of histone H3 at Lys4 (8-10). The targeting of CoREST to genes that are not repressed by REST suggests a role apart from neural cell fate regulation. These include growth factor independent (Gfi) target genes during erythroid differentiation, targets of carboxy-terminal binding protein (CtBP), and heat shock and pro-inflammatory response genes (11-15).
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