Western blot analysis of extracts from various cell lines using CNOT6 (E1L8F) Rabbit mAb.
Western blot analysis of extracts from 293 cells, mock transfected (-) or transfected with a construct expressing Myc/DDK-tagged full-length human CNOT6 (hCNOT6-Myc/DDK; +), using CNOT6 (E1L8F) Rabbit mAb (upper), DYKDDDDK Tag (9A3) Mouse mAb #8146 (middle), or β-Actin (D6A8) Rabbit mAb #8457 (lower).
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 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
CNOT6 (E1L8F) Rabbit mAb recognizes endogenous levels of total CNOT6 protein.Species Reactivity:
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Gly400 of human CNOT6 protein.
The evolutionarily conserved CCR4-NOT (CNOT) complex regulates mRNA metabolism in eukaryotic cells (1). This regulation occurs at different levels of mRNA synthesis and degradation, including transcription initiation, elongation, deadenylation, and degradation (1). Multiple components, including CNOT1, CNOT2, CNOT3, CNOT4, CNOT6, CNOT6L, CNOT7, CNOT8, CNOT9, and CNOT10 have been identified in this complex (2). In addition, subunit composition of this complex has been shown to vary among different tissues (3).
The various CNOT proteins display different functions, with CNOT6, CNOT6L, CNOT7 and CNOT8 exhibiting 3'-5' RNAse activity (2,4). Research studies indicate that the CCR4-NOT deadenylase subunits CNOT6 and CNOT6L help mediate cell survival and play a role in cell proliferation, P-body formation, and regulation of gene expression (5). Additional research evidence suggests that CNOT6 can act as a nuclear hormone receptor coactivator that associates with ZNF335 (NIF-1) to regulate expression from specific RARα regulated genes (6).
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