Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with constructs expressing Myc/DDK-tagged full-length human NF-κB p65 (hNF-κB p65-Myc/DDK; +) and Myc/DDK-tagged full-length human SETD6 (hSETD6-Myc/DDK; +), using Methyl-NF-κB p65 (Lys310) Antibody (upper) or DYKDDDDK Tag Antibody #2368 (lower).
Western blot analysis of extracts from HT-1080 and OVCAR8 cell lines using Methyl-NF-κB p65 (Lys310) Antibody. Antibodies were competed with peptides against methyl-NF-κB p65 (Lys310) (middle) or non-methyl-NF-κB p65 (Lys310) (right).
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
Methyl-NF-κB p65 (Lys310) Antibody recognizes endogenous levels of NF-κB p65 protein only when methylated at Lys310.Species Reactivity:
HumanSpecies predicted to react based on 100% sequence homology:
Mouse, Rat, Monkey, Bovine, Pig
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Lys310 of human NF-κB p65 protein. Antibodies are purified by protein A and peptide affinity chromatography.
Transcription factors of the nuclear factor κB (NF-κB)/Rel family play a pivotal role in inflammatory and immune responses (1,2). There are five family members in mammals: RelA, c-Rel, RelB, NF-κB1 (p105/p50), and NF-κB2 (p100/p52). Both p105 and p100 are proteolytically processed by the proteasome to produce p50 and p52, respectively. Rel proteins bind p50 and p52 to form dimeric complexes that bind DNA and regulate transcription. In unstimulated cells, NF-κB is sequestered in the cytoplasm by IκB inhibitory proteins (3-5). NF-κB-activating agents can induce the phosphorylation of IκB proteins, targeting them for rapid degradation through the ubiquitin-proteasome pathway and releasing NF-κB to enter the nucleus where it regulates gene expression (6-8). NIK and IKKα (IKK1) regulate the phosphorylation and processing of NF-κB2 (p100) to produce p52, which translocates to the nucleus (9-11).
Methylation of NF-κB p65 by the methyltransferase SETD6 at Lys310 results in attenuated NF-κB signaling (12).
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